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Pharmacokinetics of plasmid DNA

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Title:
Pharmacokinetics of plasmid DNA
Creator:
Houk, Brett Edward, 1969-
Publication Date:
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English
Physical Description:
xv, 165 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Administered dose ( jstor )
DNA ( jstor )
Dosage ( jstor )
Drug design ( jstor )
Liposomes ( jstor )
Pharmacokinetics ( jstor )
Plasmas ( jstor )
Plasmids ( jstor )
Rats ( jstor )
Standard deviation ( jstor )
DNA -- pharmacokinetics ( mesh )
Department of Pharmaceutics thesis Ph.D ( mesh )
Dissertations, Academic -- College of Pharmacy -- Department of Pharmaceutics -- UF ( mesh )
Plasmids -- pharmacokinetics ( mesh )
Research ( mesh )
City of Madison ( local )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 2000.
Bibliography:
Bibliography: leaves 159-165.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Brett Edward Houk.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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PHARMACOKINETICS OF PLASMID DNA


By

BRETT EDWARD HOUK










A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2000































Copyright 2000

by

Brett Edward Houk

























This work is dedicated to my parents Nancy and Ronald Houk for all of their guidance
throughout my life.














ACKNOWLEDGMENTS

I would like to acknowledge Dr. Jeffrey A. Hughes who, aside from my parents,

has been the biggest influence in my life thus far. I would also like to acknowledge Dr.

Guenther Hochhaus for his invaluable insight and guidance in this work.













TABLE OF CONTENTS
page


ACKNOW LEDGM ENTS ............................................................................................ iv

LIST OF TABLES ............................................................................................................ vii

LIST OF FIGURES ........................................................................................................ ix

ABSTRACT ..................................................................................................................... xiv

INTRODUCTION .............................................................................................................. 1

The Use of Naked pDNA as a Therapeutic Agent .................................................... 2
Effectiveness of Naked Plasmid DNA after Local Administration .................... 3
Effectiveness of Naked Plasmid DNA after IV Administration ......................... 6
Anatomical Factors Potentially Involved in the Pharmacokinetics of Plasmid DNA.. 9
Degradation of pDNA in the Bloodstream ............................................................ 16
Pharm acokinetics of Liposom al Delivery Vehicles ............................................... 17
Pharmacokinetics of Naked Plasmid DNA and Liposome: Plasmid DNA Complexes
................................................................................. ......................................... ......... 17
Pharmacokinetics of Naked Plasmid DNA in the Blood after IV Injection ......... 17
Distribution of Plasmid DNA in Tissues after IV Injection ............................. 20
Conclusions ................................................................................................................. 21

PHARMACOKINETICS OF PLASMID DNA IN ISOLATED RAT PLASMA ..... 23

Introduction ................................................................................................................. 23
M ethods ....................................................................................................................... 25
Theoretical .................................................................................................................. 35
R esu lts ......................................................................................................................... 3 6
Conclusions ................................................................................................................. 46

PHARMACOKINETICS OF PLASMID DNA AFTER IV BOLUS ADMINISTRATION
IN THE RAT ..................................................................................................................... 52

Introduction ................................................................................................................. 52
M ethods ....................................................................................................................... 53
Theoretical .................................................................................................................. 57
R esu lts ......................................................................................................................... 6 0
Conclusions ................................................................................................................. 61








DOSE DEPENDENCY OF PLASMID DNA PHARMACOKINETICS .................... 71

Introduction ................................................................................................................. 71
M ethods ....................................................................................................................... 73
Results ......................................................................................................................... 78
Conclusions ................................................................................................................. 90

PHARMACOKINETIC MODELING OF PLASMID DNA AFTER IV BOLUS
ADM IN ISTRA TION IN TH E RA T ............................................................................... 102

Introduction ............................................................................................................... 102
Theoretical ................................................................................................................ 104
Results ....................................................................................................................... 106
Conclusions ............................................................................................................... 114

PHARMACOKINETICS OF LIPOSOME: PLASMID DNA COMPLEXES .............. 122

Introduction ............................................................................................................... 122
M ethods ..................................................................................................................... 125
Results ....................................................................................................................... 127
Pharmacokinetics of Liposome:pDNA Complex Degradation in Rat Plasma ... 127
Pharmacokinetics of Liposome:pDNA Complex Degradation in Rat Whole Blood
............................................................................................................................. 12 8
Pharmacokinetics of Liposome:pDNA Complexes after IV Bolus Administration
in the Rat ........................................................................................................ 129
Conclusions ............................................................................................................... 130

CON CLU SION S AND IM PLICATION S ...................................................................... 145

Sum m ary of Results .................................................................................................. 145
Implications of Plasmid DNA Degradation in Isolated Plasma .......................... 145
Com parison of In Vitro and In Vivo Pharm acokinetics ...................................... 146
Effects of Increasing Dose of Plasm id DN A ...................................................... 148
Results of the Curve Fitting Experim ents ........................................................... 151
Liposom e: pDN A Com plex Conclusions ........................................................... 152
Future Directions ...................................................................................................... 155
Concluding Rem arks ................................................................................................. 157

LIST OF REFEREN CES ................................................................................................ 159

BIOGRA PHICA L SKETCH .......................................................................................... 166














LIST OF TABLES


Table pag

2-1. Method parameters for pDNA analysis ................................................................... 34

2-2. Pharmacokinetic parameters for pDNA in isolated rat plasma ................................ 41

2-3. Noncompartmental parameters for pDNA after incubation in isolated plasma ..... 42

2-4. Pharmacokinetic parameters for pDNA after incubating the pGE150 plasmid in
isolated rat plasm a ................................................................................................. 49

3-1. Pharmacokinetic parameters calculated after 500 .tg dose of SC pDNA ................. 65

3-2. Comparison of in vivo and in vitro pharmacokinetic parameters for pDNA ............ 66

3-3. Comparison of OC and L pDNA parameters after administration of SC pGL3,
pG E 150, and pG eneM ax ...................................................................................... 69

4-1. Pharmacokinetic parameters estimated for supercoiled pDNA based upon the fit t=0
concentration of SC pDN A ................................................................................... 81

4-2. Noncompartmental analysis of OC pDNA after IV bolus administration of SC
p D N A ......................................................................................................................... 84

4-3. Noncompartmental analysis of L pDNA after IV bolus administration of SC pDNA..85

4-4. Noncompartmental analysis of OC pDNA after IV bolus administration of OC
pDNA at 2500 and 250 tg doses .......................................................................... 89

4-5. Noncompartmental analysis of L pDNA after IV bolus administration of L pDNA at
2500 and 250 tg doses .......................................................................................... 93

5-1. Pharmacokinetic parameters for pDNA based upon the model presented in the text... 112

5-2. Overall pharmacokinetic parameters for pDNA when all doses are fit
sim ultan eously ........................................................................................................... 113








5-3. Pharmacokinetic parameters calculated after administration of OC pDNA at 2500
and 250 g g doses ........................................................................................................ 118

5-4. Pharmacokinetic parameters calculated after administration of L pDNA at 2500 and
250 ptg doses .............................................................................................................. 119

6-1. Noncompartmental analysis of pDNA after administration of liposome: SC pDNA
com p lex es .................................................................................................................. 137

6-2. Comparison of SC pDNA pharmacokinetic parameters after administration of SC
pDNA either in free form (naked) at 2500 [tg dose or after administration as
liposome: pDNA complexes at 500 ptg dose ............................................................. 138

6-3. Comparison of OC pDNA pharmacokinetic parameters after administration of SC
pDNA either in free form (naked) or after administration as liposome: pDNA
com plexes at 500 jig pDN A dose .............................................................................. 139

6-4. Comparison of L pDNA pharmacokinetic parameters after administration of SC
pDNA either in free form (naked) or after administration as liposome: pDNA
com plexes at 500 jtg pD N A dose .............................................................................. 140














LIST OF FIGURES


Figure p

1-1. Potential sights for nicking of the phosphodiester backbone of DNA ...................... 10

1-2. Model of plasmid DNA degradation in the bloodstream .......................................... 11

1-3. Schematic representation of pDNA (.) passing through a continuous capillary: (1)
pinocytosis, (2) through intercellular junctions, and (3) passing through
endothelial channels ............................................................................................... 12

1-4. Schematic representation of pDNA (.) passing through a fenestrated capillary: (1)
pinocytosis, (2) passing through a diaphragm fenestrae, and (3) passing through
and open fenestrae ................................................................................................. 13

1-5. Schematic representation of pDNA (e) passing through a discontinuous capillary.
(1) pinocytosis and (2) passing through large pores in the endothelium .............. 14

2-1. Plasm id m ap of pGL3 Control ................................................................................. 26

2-2. Plasmid map of the pGeneMax-Luciferase ............................................................ 27

2-3. Plasm id m ap of pG E150 .......................................................................................... 28

2-4. Standard curve for supercoiled pDNA. Abcissa represents total ng estimated by UV
absorbance at 260 nm. Ordinate represents ng calculated by the KDS ID
software. Error bars represent 1 standard deviation .......................................... 31

2-5. Standard curve for OC pDNA. Abcissa represents total ng estimated by UV
absorbance at 260 nm. Ordinate represents ng calculated by the KDS ID
software. Error bars represent 1 standard deviation .......................................... 32

2-6. Standard curve for Linear pDNA. Abcissa represents total ng estimated by UV
absorbance at 260 nm. Ordinate represents ng calculated by the KDS ID
software. Error bars represent 1 standard deviation .......................................... 33

2-7. Pharmacokinetic model of plasmid DNA degradation in rat plasma. The model is
considered to be a unidirectional process. SC, OC, and L represent the amounts
of supercoiled, open circular, and linear plasmid, respectively, in each








compartment. The rate constants k,, ko, and k, represent the degradation constants
for supercoiled, open circular, and linear plasmid, respectively ........................... 37

2-8. Agarose gel analysis of pDNA degradation in isolated rat plasma. Lane 1; size
standard, lane 2; 30 sec, lane 3; 1 min, lane 4; 2 min, lane 5; 3 min, lane 6; 5 min,
lane 7; 10 min, lane 8; 20 min, lane 9; 30 min, lane 10; 45 min, lane 11; 60 min,
lane 12; 80 m in ...................................................................................................... 39

2-9. Experimental and fitted data based on the pharmacokinetic model described in the
text. Data points represent actual experimental data. Lines represent values
predicted by the model. Data represents mean of n-3 1 standard deviation.
Key: U supercoiled, open circular, A linear .................................................. 40

2-10. Analysis of rate constants in dilute plasma. Degradation rate constants were
modeled in PBS diluted rat plasma. Rate constants represent the fitted values of
n=6 rats/ time point. Key: *ks in dilute plasma, Sk0 in dilute plasma. The value
of k, is not reported due to the prolonged stability of linear plasmid in dilute
p lasm a ........................................................................................................................ 44

2-11. Degradation of supercoiled pDNA in 25% rat plasma after (A) incubating the
plasma at 90'C for 10 min (B) the addition of 0.1 mM EDTA ............................ 45

2-12. Comparison of concentrations of OC pDNA using (*) pGE150 concentrations of
OC pDNA using (0) pGL3 in isolated rat plasma. Data represents mean of n=3
1 standard deviation ............................................................................................. 47

2-13. Comparison of concentrations of L pDNA using (*) pGE 150 versus
concentrations of L pDNA using (0) pGL3 in isolated rat plasma. Data
represents mean of n=3 1 standard deviation ..................................................... 48

3-1. Photograph of the jugular cannula placement used for blood sampling ................... 55

3-2. Photograph of the femoral vein isolation and injection procedure used for IV bolus
adm inistration ............................................................................................................ 56

3-3. A representative gel from which plasmid amounts were quantified as described in
the methods section. Lane 1: size standard, lane 2: 1 min, lane 3: 2 min, lane 4:
3.5 min, lane 5: 5 min, lane 6:10 min, lane 7: 20 min, lane 8: 30 min, lane 9: 45
m in, lane 10: 60 m in ............................................................................................ 59

3-4. Experimental and fitted data based on the pharmacokinetic model described in the
text. Data points represent actual experimental data. Lines represent values
predicted by the model. Data represents mean of n=6 I standard devaition.
K ey: 0 open circular, A linear ............................................................................ 62

3-5. Concentrations of OC pGL3 after 0: IV bolus administration of a 500 [-tg dose of
SC pGL3, and *: Incubation of SC pGL3 in isolated plasma at 37'C ................. 63








3-6. Concentrations of L pGL3 after 0: IV bolus administration of a 500 jIg dose of SC
pGL3, and *: Incubation of SC pGL3 in isolated plasma at 37'C ..................... 64

3-7. Concentrations of OC pDNA in the bloodstream after IV bolus administration of
500 [tg of pCMV-Luc, pGE150, or pGL3. Data represents mean of n=3 I
standard deviation ................................................................................................. 67

3-8. Concentrations of L pDNA in the bloodstream after IV bolus administration of 500
jig of pCMV-Luc, pGE150, or pGL3. Data represents mean of n=3 1 standard
d ev iation ..................................................................................................................... 6 8

4-1 Agarose gel analysis of pDNA after conversion to the OC form of the plasmid. Lane
1: Prior to treatment plasmid is predominately SC. Lane 2: After treatment
plasmid is completely converted to to OC form ................................................... 75

4-2. Absorbance of pDNA before and after conversion to the OC form. Data represents
averages of n=3 I standard deviation ............................................................... 76

4-3. Agarose gel analysis of pDNA before and after conversion to the L form of the
plasmid. Lane 1: Size standard, Lane 2: before treatment the plasmid is
predominately in the SC and OC form, Lane 3: Mixture of SC, OC, and L
plasmid for reference, Lane 4: after treatment the plasmid is completely converted
to th e L form .............................................................................................................. 77

4-4. Concentrations of SC pDNA in the bloodstream after 2500 jig dose. SC pDNA
remained detectable through 1 minute after administration. Data points represent
averages of n=3 1 standard deviation. Lines represent a least squares fit of the
data using the model described in the Methods section ........................................ 80

4-5. Concentrations of OC pDNA after IV bolus administration of: U 2500 jig, A 500
pg, O 333 jig, or 250 jig of SC pDNA. Data represents mean of n=3 ............ 82

4-6. Concentrations of L pDNA after IV bolus administration of: N 2500 pig, A 500 [1g,
333 pjg, or 250 jig of SC pDNA. Data represents mean of n=3 .................. 83

4-7. Superposition of OC pDNA concentrations normalized for dose after administration
of 0: 2500 jig, A: 500 jig, +: 333 jig, or 0:250 jtg dose. Data represents mean
of n=3 I standard deviation .............................................................................. 86

4-8. Concentrations of OC pDNA in the bloodstream after administration of OC pDNA
at a 2500 jig dose. Data represents mean of n=3 1 standard deviation ............. 87

4-9. Concentrations of OC pDNA in the bloodstream after administration of OC pDNA
at a 250 pg dose. Data represents mean of n=3 I standard deviation ............... 88

4-10. Concentrations of L pDNA in the bloodstream after administration of L pDNA at a
2500 jig dose. Data represents averages of n=3 1 standard deviation .............. 91








4-11. Concentrations of L pDNA in the bloodstream after administration of L pDNA at a
250 jag dose. Data represents averages of n=3 1 standard deviation ................ 92

4-12. Area under the curve of OC pDNA after administration of a 2500 jIg dose of SC or
OC pDNA. Data represents mean of n-3 1 standard deviation. Indicates
statistical significance by one way ANOVA (p<0.05) ....................................... 94

4-13. Area under the curve of OC pDNA after administration of a 250 jig dose of SC or
OC pDNA. Data represents mean of n=3 1 standard deviation. Indicates
statistical significance by one way ANOVA (p<0.05) ......................................... 95

4-14. Area under the curve of L pDNA after administration of a 2500 jig dose of SC or
OC pDNA. Data represents mean of n=3 1 standard deviation. Indicates
statistical significance by one way ANOVA (p<0.05) ......................................... 96

4-15. Area under the curve of L pDNA after administration of a 250 jig dose of SC or
OC pDNA. Data represents mean of n=3 1 standard deviation. AUC
differences were not statistically significant by one way ANOVA ...................... 97

5-1. M odel for pDNA clearance from the bloodstream ........................................................ 105

5-2. Concentrations of(A) OC and (B) L pDNA in the bloodstream after 2500 lag dose
of SC pDNA. Data points represent the averages of n=3 1 standard deviation.
Lines represent concentrations predicted by the model ............................................. 108

5-3. Concentrations of(A) OC and (B) L pDNA in the bloodstream after 500 jig dose of
SC pDNA. Data points represent the averages of n=3 1 standard deviation.
Lines represent concentrations predicted by the model ............................................. 109

5-4. Concentrations of(A) OC and (B) L pDNA in the bloodstream after 333 jig dose of
SC pDNA. Data points represent the averages of n=3 1 standard deviation.
Lines represent concentrations predicted by the model ............................................. 110

5-5. Concentrations of (A) OC and (B) L pDNA in the bloodstream after 250 jig dose of
SC pDNA. Data points represent the averages of n=3 1 standard deviation.
Lines represent concentrations predicted by the model ............................................. 111

5-6. Concentrations of OC pDNA in the bloodstream after (A) 2500 jig and (B) 250 jig
dose of OC pDNA. Data points represent the averages of n=3 1 standard
deviation. Lines represent concentrations predicted by the model ........................... 115

5-7. Concentrations of L pDNA in the bloodstream after (A) 2500 jig and (B) 250 jig
dose of OC pDNA. Data points represent the averages of n=3 1 standard
deviation. Lines represent concentrations predicted by the model ........................... 116








5-8. Concentrations of L pDNA in the bloodstream after (A) 2500 jIg and (B) 250 jIg
dose of L pDNA. Data points represent the averages of n=3 1 standard
deviation. Lines represent concentrations predicted by the model ........................... 117

6-1. Liposome-pDNA complexes were incubated in rat plasma for various time points.
10 j.1 of sample was loaded in each lane as described in the methods section.
Lane 1; size standard, lane 2; 1 min, lane 3; 2 min, lane 4; 5 min, lane 5; 10 min,
lane 6; 20 min, lane7; 30 min, lane 8; 60 min, lane9; 2 h, lane 10; 3 h, lane 11; 5.5
h .................................................................................................................................. 13 1

6-2. Agarose gel analysis of liposome/pDNA complexes. (A) 1:1 lipid:pDNA ratio,
through 4 hours. (B) 3:1 lipid:pDNA ratio, through 6 hours. (C) 6:1 lipid:pDNA
ratio, through 6 hours. *Indicates the 3 hour time point ........................................... 132

6-3. Lane 1: high molecular weight size standard, lane 2:1:1 lipid:pDNA complexes,
lane 3: 3:1 lipid:pDNA ratio (w/w), lane 4: 6:1 lipid:pDNA ratio ............................ 133

6-4. (A)Degradation of SC pDNA in rat blood versus plasma. (B)Degradation of
supercoiled pDNA in 3:1 and 6:1 (w/w) liposome/pDNA complexes incubated in
heparinized rat whole blood. Error bars indicate standard deviation of n=3 rats .... 134

6-5. Agarose gel analysis of pDNA after administration of liposome: pDNA complexes.
Lane 1: 15 sec, lane 2: 30 sec, lane 3: 45 sec, lane 4: 1 min, lane 5: 1.5 min, lane
6:2 min, lane 7:2.5 min, lane 8:3 min, lane 9:4 min, lane 10:5 min ..................... 135
6-6. Plasma concentrations of SC, OC, and L pDNA after 500 jig IV bolus

administration of SC pDNA: liposome complexes. Key: *: SC, U: OC, A: L ....... 136

7-1. Schematic representation of pDNA degradation in isolated plasma ............................. 147

7-2. Schematic representation of pDNA pharmacokinetic parameters after IV bolus
adm inistration of SC pDN A in the rat ....................................................................... 154

7-3. Schematic representation of liposome pDNA clearance from the bloodstream. In
this model, removal from the bloodstream of the lipid: pDNA complexes is
assumed to be larger than the degradation of the complex ........................................ 156














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

PHARMACOKINETICS OF PLASMID DNA

By

Brett E. Houk

May 2000

Chairman: Dr. Jeffrey A. Hughes
Cochairman: Dr. Guenther Hochhaus
Major Department: Pharmaceutics

We sought to construct a complete pharmacokinetic model to describe the

degradation of all three topoforms, supercoiled (SC), open circular (OC), and linear (L),

of pDNA in vivo and in vitro. SC pDNA was incubated in isolated rat plasma at 37C in

vitro. At various time points, the plasma was assayed by electrophoresis for the amounts

of SC, OC, and L pDNA remaining. The calculated amounts remaining were fit to linear

differential equations describing this process. The calculated pharmacokinetic

parameters suggested that SC pDNA degrades in isolated rat plasma with a half-life of

1.2 min, OC pDNA degrades with a half-life of 21 min, and L pDNA degrades with a

half-life of 11 min. Complexation of pDNA with cationic liposomes resulted in a portion

of the supercoiled plasmid remaining detectable through 5.5 h in vitro. We next

investigated the pharmacokinetics of SC plasmid DNA after IV bolus administration in

the rat by following SC, OC, and L pDNA. SC pDNA was detectable in the bloodstream

only after the highest, 2500 tg, dose and had a clearance of 390(10) ml/min and volume








of distribution of 148(26) ml. The pharmacokinetics of OC pDNA exhibited non-linear

characteristics with clearance ranging from 8.3(0.8) to 1.3(0.2) ml/min and a volume

of distribution of 39(19) ml. L pDNA exhibited linear kinetics and was cleared at

7.6(2.3) ml/min with a volume of distribution of 37(17) ml. AUC analysis revealed

60(10) % of the SC was converted to the OC form, and nearly complete conversion of

the OC pDNA to L pDNA. Clearance of SC pDNA was decreased after liposome

complexation to 87(30) ml/min. However, the clearance of OC and L pDNA was

increased relative to naked pDNA at an equivalent dose to 37(9) ml/min and 95(37)

m/min, respectively. We conclude that SC pDNA is rapidly cleared from the circulation.

OC pDNA displays non-linear pharmacokinetics. L pDNA exhibits first order kinetics.

Liposome complexation protects the SC topoform, but the complexes are more rapidly

cleared than the naked pDNA.













CHAPTER 1
INTRODUCTION

Biotechnology is one of the most rapidly growing areas in the pharmaceutical

sciences today. However, biotechnology products (e.g. proteins and peptides) suffer

from poor stability, low absorption, and difficulties in delivery. It would therefore be

ideal if the protein could be made in vivo, utilizing the body's own mechanisms to

produce the competent protein. Gene therapy is one potential route by which to

accomplish this goal. Gene therapy also offers the potential treatment of genetic

diseases. The replacement of mutated, missing, or deleted DNA via gene therapy can

result in the production of a competent protein. These potentials make gene therapy one

of the most exciting and rapidly advancing areas of biotechnology.

Early studies have revealed that systemically administered plasmid DNA (pDNA)

can be expressed in animals (Kawabata et al. 1995; Mahato et al. 1995; Osaka et al. 1996;

Song et al. 1997; Thierry et al. 1997) and humans (Valere 1999). Intravenous (IV)

administration of DNA offers the potential advantage of allowing a wide distribution of

activity in the body (Lew et al. 1995; Thierry 1995; Osaka et al. 1996; Thierry et al.

1997). This route of administration allows the treatment of non-localized and systemic

diseases. Previous research on the pharmacokinetics of non-viral gene therapies have

only been observational citing that plasmid DNA degrades within 5 minutes after

incubation in whole blood in vitro or after IV injection (Kawabata et al. 1995; Thierry et

al. 1997).








Plasmid DNA exists as three major topoforms. The native structure of non-

damaged pDNA is supercoiled (SC). Single strand nicks to the phosphodiester backbone

of pDNA yield an open circular (OC) form (Figure 1-1). This metabolite of SC pDNA is

associated with significant transcriptional activity (-90-100%) (Adami et al. 1998; Niven

et al. 1998). Further single strand nicks to the OC pDNA yield linear (L) pDNA,

associated with a significant loss of activity (-90%). This process is schematically

illustrated in Figure 1-2.

In order to properly dose and achieve the desired levels of gene expression it will

be necessary to understand the pharmacokinetics of pDNA. In initial human clinical

trials with viral gene therapy, at least one study was terminated due to a patient death

(Press 1999). This death was later attributed to the high doses utilized in the trails. Thus,

the pharmacokinetics of pDNA is an essential area to be considered as gene therapy

approaches clinical use.

The Use of Naked pDNA as a Therapeutic Agent

The use of naked pDNA as a drug after intravenous (IV) administration has been

intensely investigated (Wang et al. 1995; Takeshita et al. 1996; Zhang et al. 1997; Budker

et al. 1998; Song et al. 1998; Wang et al. 1998; Witzenbichler et al. 1998; Liu 1999;

Zhang et al. 1999). The use of naked pDNA in vivo was initially reported after

intramuscular (IM) or intradermal (SQ) administration in mammals (Wolff et al. 1991;

Fazio et al. 1994; Katsumi et al. 1994; Bright et al. 1995; Donnelly et al. 1995; Lopez-

Macias et al. 1995; Ulmer et al. 1995; Bright et al. 1996; Corr et al. 1996; Casares et al.

1997; Danko et al. 1997; Lawson et al. 1997; Ragno et al. 1997; Haensler et al. 1999;

Noll et al. 1999; Osorio et al. 1999). These studies have definitively shown efficient

expression of a transgene can be achieved after administration of naked pDNA. The








successes in these studies suggest that pharmacokinetic modeling of pDNA in the

bloodstream after IV administration, or pDNA appearing in the bloodstream after local

administration, is an area that must be more clearly defined in order to optimize gene

therapy for clinical use.

Effectiveness of Naked Plasmid DNA after Local Administration

Fazio and coworkers (Fazio et al. 1994) demonstrated that a transgene could be

efficiently secreted into the circulation after IM administration. Plasma accumulation of

human Apo-E2 was demonstrated for at least 45 days after injection. After

administration of pDNA encoding for an interferon transgene, interferons were detected

from days 7 to 28 post-DNA innoculation (Lawson et al. 1997). Administration of

plasmid DNA encoding the chloramphenicol acetyltransferase gene (CAT) in sterile

water lead to CAT transgene expression that peaked between 1 and 3 days and was

detected up to 28 days after DNA administration. Together these results indicate that

sustained expression can be obtained.

Efficient immunization of monkeys, mice, dogs, and cats has been demonstrated

using naked pDNA (Katsumi et al. 1994; Lopez-Macias et al. 1995; Ulmer et al. 1995;

Bright et al. 1996; Ragno et al. 1997; Haensler et al. 1999; Noll et al. 1999; Osorio et al.

1999). After injection of naked pDNA encoding for influenza hemagglutinin into the

skin of mice and monkeys, induction of significant ELISA antibody titers and

hemagglutination (HA) inhibition titers that were above the usual threshold values

predictive of protection against influenza were demonstrated (Haensler et al. 1999). Mice

immunized by various mucosal routes with a pDNA carrying the HA gene (pVlj- HA)

induced a HA-specific cytotoxic T lymphocyte (CTL) response. Similarly, nasal








immunization with the DNA vaccine induced primary CTLs against measles virus HA

(Etchart et al. 1997).

Plasmid DNA may also serve as an attractive means by which immunization to

parasitic infection may be achieved. After injection of pDNA encoding for heat shock

protein 65, T cell proliferation and antibodies to this protein were found to be elevated in

rats when compared with both an arthritic control and nafve animals (Ragno et al. 1997).

A single immunization with pDNA encoding for Yersinia enterocolitica 60-kDa heat

shock protein (Y- HSP60) was used for vaccination and induced significant Y-HSP60-

specific T cell responses after 1 week (Noll et al. 1999). Induction of antibodies against

Salmonella typhi OmpC porin by naked DNA immunization has also been demonstrated

(Lopez-Macias et al. 1995).

A pDNA expression vector encoding human factor IX as an example of

immunogen was injected into mice three times at 10-day intervals (Katsumi et al. 1994).

This resulted in production of antibodies to human factor IX. Spleen cells from

inoculated mice also showed significant cytotoxic T lymphocyte response to target cells

expressing human factor IX. Thus, IM and SQ injection of pDNA can induce immune

responses against the encoded protein without an exposure to virus particles, and this

approach may serve as the basis for immunotherapy in the treatment of cancer and

infectious diseases in humans.

Plasmid DNA encoding for viral proteins is also an attractive means by which

immunization to viral infection may be achieved. The applicability of pDNA

immunization technology for vaccine development was also investigated by immunizing

dogs and cats by the IM and SQ routes with a pDNA vector encoding the rabies virus








glycoprotein G (Osorio et al. 1999). The results demonstrated that non-facilitated, naked

pDNA vaccines can elicit strong, antigen-specific immune responses in dogs and cats,

and DNA immunization may be a useful tool for future development of novel vaccines

for these species. Plasmid DNA encoding for the large tumor antigen (T- Ag) of SV40

was used to actively immunize mice to assess the induction of SV40 T-Ag-specific

immunity (Bright et al. 1996). Direct injection of the recombinant SV40 T-Ag protein

alone failed to induce SV40 T-Ag-specific CTL responses, whereas the pDNA encoding

SV40 T-Ag elicited CTL activity specific for SV40 T-Ag. Naked pDNA induced

immune responses that were protective against a lethal challenge with SV40-transformed

cells.

Naked pDNA has also been successful in the treatment of cancer by local

administration. Direct intratumoral injection of free pDNA into mouse melanoma BL6

solid tumor can also result in a high level of transfection. The average amount of

chloramphenicol acetyltransferase (CAT) expressed by injecting 30 Ig pDNA containing

a CAT gene into a single BL6 tumor was 1.9 +/- 1.0 ng, which is comparable to that

reported in the skeletal muscle (Yang and Huang 1996). An intratumoral injection of

naked pDNA containing the HSV-TK gene (pAGO) resulted in tumor weight reduction

(40-50%) in treated animals versus control groups. Moreover, histopathological analysis

on tumors showed large areas of cavitary necrosis (85%) in treated groups compared to

controls (10%) (Soubrane et al. 1996). Thus direct injection of free pDNA may offer a

simple and effective approach and might be a potential method for cancer gene therapy.








Effectiveness of Naked Plasmid DNA after IV Administration

Naked pDNA administration by IV injection has also been shown to be an

effective means by which high levels of gene expression can be obtained (Wang et al.

1995; Takeshita et al. 1996; Zhang et al. 1997; Budker et al. 1998; Song et al. 1998;

Wang et al. 1998; Witzenbichler et al. 1998; Liu 1999; Zhang et al. 1999). Budker and

coworkers demonstrated that pDNA can be delivered to and expressed within skeletal

muscle of rats when injected rapidly, in a large volume (2 to 3 ml) (Budker et al. 1998).

Liu and coworkers also showed naked pDNA can be efficiently expressed in mice

(Liu 1999). As high as 45 tg of luciferase protein per gram of liver could be recovered

by a single tail vein injection of 5 ig of naked pDNA. Approximately 45% of

hepatocytes expressed the transgene. Peak expression was obtained at 8 hours after

administration and could be retained with repeated injections.

Efficient naked pDNA expression has been obtained following delivery via the

portal vein, hepatic vein, bile duct or direct IV administration via the tail vein in mice,

rats, and dogs (Zhang et al. 1997; Zhang et al. 1999). The highest levels of expression

were achieved after IV administration by rapidly injecting the pDNA in large volumes,

approximately 2.5 ml. Over 15 jig of luciferase protein/liver was produced in mice and

over 50 gg in rats. Equally high levels of beta-galactosidase (beta-Gal) expression were

obtained, in over 5% of the hepatocytes that had intense blue staining. Expression of

luciferase or beta-Gal was evenly distributed in hepatocytes throughout the entire liver

when either of the three routes were injected. Peri-acinar hepatocytes were preferentially

transfected when the portal vein was injected in rats. These levels of foreign gene

expression are among the highest levels obtained with nonviral vectors. Repetitive








pDNA administration through the bile duct led to sustianed foreign gene expression.

This study demonstrates that high levels of pDNA expression in hepatocytes can be

easily obtained by IV injection.

Takeshita and coworkers (Takeshita et al. 1996) investigated the hypothesis that

naked pDNA encoding for vascular endothelial growth factor (VEGF) could be used in a

strategy of arterial gene therapy to stimulate collateral artery development. Plasmid

DNA encoding each of the three principle human VEGF isoforms (phVEGF121,

phVEGF165, or phVEGF189) was applied to the hydrogel polymer coating of an

angioplasty balloon and delivered percutaneously to one iliac artery of rabbits with

operatively induced hindlimb ischemia. Compared with control animals transfected with

LacZ, site-specific transfection of phVEGF resulted in augmented collateral vessel

development documented by serial angiography, improvement in calf blood pressure

ratio (ischemic to normal limb), resting and maximum blood flow, and capillary to

myocyte ratio (suggesting increased vascularization). Similar results were obtained with

phVEGF121, phVEGF165, and phVEGF189. This suggests that these isoforms are

biologically equivalent with respect to in vivo angiogenesis. The potential for VEGF-C

to promote angiogenesis in vivo was then tested in a rabbit ischemic hindlimb model

(Witzenbichler et al. 1998). Ten days after ligation of the external iliac artery, VEGF-C

was administered as naked pDNA (pcVEGF-C; 500 [ig) from the polymer coating of an

angioplasty balloon or as recombinant human protein (rhVEGF-C; 500 [tg) by direct

intra-arterial infusion. Physiological and anatomical assessments of angiogenesis 30 days

later showed evidence of therapeutic angiogenesis for both pcVEGF-C and rhVEGF-C.

Hindlimb blood pressure ratio (ischemic/normal) after pcVEGF-C increased after








pcVEGF-C versus controls and after rhVEGF-C versus control rabbits receiving rabbit

serum albumin. Doppler- derived iliac flow reserve was increased for pcVEGF-C versus

controls and increased for rhVEGF-C versus albumin controls. Neovascularity was

documented by angiography in vivo after administration of pcVEGF-C and capillary

density was measured at necropsy increased. Arterial gene transfer of naked pDNA

encoding for a secreted angiogenic cytokine, thus, represents a potential alternative to

recombinant protein administration for stimulating collateral vessel development.

Naked pDNA constructs encoding for the human kallikrein protein delivered to

spontaneously hypertensive rats via IV injection have been shown to be efficient at

controlling hypertension (Wang et al. 1995). The expression of human tissue kallikrein in

rats was identified in the heart, lung, and kidney by reverse transcription polymerase

chain reaction followed by Southern blot analysis and an ELISA specific for human

tissue kallikrein. A single injection of both human kallikrein pDNA constructs caused a

sustained reduction of blood pressure, which began 1 week after injection and continued

for 6 weeks. A maximal effect of blood pressure reduction of 46 mm Hg in rats was

observed 2-3 weeks after injection with kallikrein pDNA as compared to rats with vector

pDNA. These results show that direct gene delivery of human tissue kallikrein causes a

sustained reduction in systolic blood pressure in genetically hypertensive rats and

indicate that the feasibility of kallikrein gene therapy for treating human hypertension

should be studied.

Collectively, these results suggest that IV administration of naked pDNA is an

attractive means to treat a large range of diseases. However, complete pharmacodynamic

modeling of pDNA will has not been achieved. This will allow correlation of the








administered dose with the desired levels of gene expression at the site of activity.

Because of the plasmids high molecular weight, anatomical factors must be considered in

the movement of these molecules within the body.

Anatomical Factors Potentially Involved in the Pharmacokinetics of Plasmid DNA

Plasmid DNA is a macromolecule having a molecular weight of 3.5 million for a

typical plasmid of 5.5 kilobase pairs. This large molecular weight results in an increased

likelihood of clearance processes being a function of size and its resulting abitily to pass

through capillary endothelia. After IV administration, distribution of macromolecules is

limited by the structure of the vascular endothelium. The structure of capillaries is

diverse among organs. There are 3 main types of blood capillaries: continuous,

fenestrated, and discontinuous (Hwang et al. 1997; Takakura et al. 1996).

These 3 types of capillaries are represented in Figures 1-3, 1-4, and 1-5. The

diameter of the free plasmid varies from between 8 to 22 rum (Yarmola 1985). The

passage of pDNA through a continuous capillary would be limited to the 50 nm

pinocytotic vesicles, 2 to 6 nm intracellular junctions, and 50 nm transendothelial

channels (Figure 1-3) (Hwang et al. 1997). The basal lamina presents a barrier of

collagen, glycoproteins, and fibronectin, macromolecules greater than 11 Im can be

retained by the basal lamina. Thus, this may present a barrier for diffusion of the plasmid

(Hwang et al. 1997). Continuous capillaries are the most widely distributed in

mammalian tissue and are found in skeletal, cardiac, and smooth muscles, as well as lung,

skin, subcutaneous tissues, serous membranes, and mucus membranes (Takakura et al.

1996).











































Figure 1-1. Potential sights for nicking of the phosphodiester backbone of DNA.

























Endonuclease action: Endonuclease action:
Single strand nick to Single strand nick
the plasmid adjacent to previous


Endonuclease or
exonuclease action


Figure 1-2. Model of plasmid DNA degradation in the bloodstream.


10'





















0
50 nm


2
2-6 nm


0
50 nm


---I


Figure 1-3. Schematic representation of pDNA (.) passing through a continuous
capillary: (1) pinocytosis, (2) through intercellular junctions, and (3) passing through
endothelial channels.





















50 nm


40-60 nm


40-60 nm


S S


Figure 1-4. Schematic representation of pDNA (.) passing through a fenestrated
capillary: (1) pinocytosis, (2) passing through a diaphragm fenestrae, and (3) passing
through and open fenestrae.


-j


















1


50 nm


2

0
10'-10' nm


Figure 1-5. Schematic representation of pDNA (.) passing through a discontinuous
capillary. (1) pinocytosis and (2) passing through large pores in the endothelium.


F---










Fenestrated capillaries (Figure 1-4) are more likely to allow passage of pDNA

into tissues. The pDNA may be transported through mechanisms similar to those

involved in the continuous capillary, in addition to transport through 20 to 60 ram

fenestrae (Hwang et al. 1997; Takakura et al. 1996). These fenestrae may or may not be

closed by a diaphragm. The diameter of the closed diaphragm has not been reported.

This type of capillary is generally found in the intestinal mucosa, endocrine glands,

exocrine glands, glomerulus, and peritubular capillaries (Takakura et al. 1996).

Discontinuous capillaries are characterized by endothelial gaps and large pores

with diameters ranging from 100 to 1000 nm (Figure 1-5) (Hwang et al. 1997; Takakura

et al. 1996). In these capillaries there is little restriction of diffusion of macromolecules.

Another characteristic of this type of capillary is the lack of a basal lamina (Hwang et al.

1997). The mucopolysaccharide rich interstitial Spaces of Disse have pore diameters

ranging from 36 to 50 nrm and are unlikely to present a major barrier for the transport of

pDNA. The discontinuous capillary is more limited in its distribution than the other

types and is found only in the liver, spleen, and bone marrow (Takakura et al. 1996).

These anatomical features can play an important role in the distribution of IV

administered pDNA, and other macromolecules. In addition, capillary permeability can

be further enhanced in pathophysiological states such as cancer and inflammation

(Takakura et al. 1996). Thus the fate of IV administered pDNA is determined not only

by physio-chemical properties such as molecular weight, but also by anatomical features

of the capillary endothelium present in each tissue.








Degradation of pDNA in the Bloodstream

Early studies suggested that serum nucleases play a major role in the clearance of

DNA from the bloodstream of injected animals (Gosse et al. 1965). Investigations by

Chused and coworkers suggested that nucleases may not play a major role in the

degradation of tritiated KB cell genomic DNA when IV injected in mice (Chused 1972).

However, their assay was not able to identify the true activity of nucleases given that

their assay utilized genomic DNA. Single strand cuts to the isolated genomic DNA

would not yield small fragments and would be undetectable by their method. This would

yield an underestimation of true nuclease activity. In contrast, single strand cuts to

pDNA would lead to a degradation of the native SC structure to the OC form of the

plasmid and be detectable by agarose gel analysis.

Nucleases represent two subclasses of enzymes, endonucleases and exonucleases.

Endonucleases act on the phosophodiester backbone of DNA in a continuous chain

(Lodish 1995). Whereas, endonucleases act upon the free end (5' or 3') of the

phosphodiester backbone in a linear segment of DNA. Investigations by Thierry and

coworkers, utilizing agarose gel analysis, suggested that the main nuclease activity in the

bloodstream was endonucleolytic. This was based on the finding that the linear to

supercoiled ratio increased with time and the SC: OC ratio remained identical to control

(Thierry et al. 1997). However this view fails to recognize endonucleolytic activity on

linear pDNA also generates degradation products. If endonuclease activity is the primary

route of degradation, the kinetic ratios should all remain similar, owing to the fact that

exonuclease activity would be masked by endonuclease activity. The pharmacokinetics

of this degradation remain to be determined and may serve as a valuable tool in the

understanding of the mechanisms of pDNA degradation observed in the bloodstream.








Pharmacokinetics of Liposomal Delivery Vehicles

Although few studies are available on the pharmacokinetics of liposome:pDNA

complexes, liposomal pharmacokinetics alone have been studied extensively with several

reviews published (Hwang et al. 1997; Juliano 1988; Takakura et al. 1996). Liposome

pharmacokinetics have been shown to be dependent upon size (Sato 1986), dose

(Bosworth 1982; Osaka et al. 1996), lipid composition (Gabizon 1988), and charge

(Juliano 1988). In general, liposomes larger than 60 nm in diameter are unable to access

tissues having continuous capillary endothelia, including skeletal, cardiac, and smooth

muscle, lung, skin, subcutaneous tissue, and serous and mucous membranes, and are

limited to uptake in tissues of the reticuloendothelial system (Hwang et al. 1997).

Liposomes larger than 0.5 p.m are confined to the vasculature in all tissues.

Pharmacokinetics of Naked Plasmid DNA and Liposome: Plasmid DNA Complexes

After systemic administration of pDNA alone or as liposome:pDNA complexes,

DNA rapidly disappears from the bloodstream. The processes responsible involve

degradation in the blood stream, interaction with plasma proteins, organ distribution, and

uptake by the reticuloendothelial system. The transport of DNA and liposome:pDNA

complexes into organs is roughly a unidirectional system, where distribution back into

the central compartment can be assumed to be negligible (Mahato et al. 1997).

Pharmacokinetics of Naked Plasmid DNA in the Blood after IV Injection

Plasma levels of pDNA may be measured using radiolabeled DNA or agarose gel

analysis (Kawabata et al. 1995; Lew et al. 1995; Mahato et al. 1995; Osaka et al. 1996;

Thierry et al. 1997). Using agarose gel analysis, Thierry and coworkers found that SC

plasmid DNA is not detectable in either murine plasma or cell fractions 1 minute after

injection of naked plasmid DNA in mice (Thierry et al. 1997). OC and L forms have








been detected through 30 minutes post-injection by Southern blot analysis (Lew et al.

1995). The half-life of intact (OC or L) plasmid DNA is less than 5 minutes. Degraded

plasmid fragments remain detectable in the blood at 30 minutes post injection. By 60

minutes even degraded plasmid is cleared. This elimination has been shown to be

independent of the DNA sequence (Lew et al. 1995).

When plasmid DNA was administered to mice in the form of liposome:pDNA

complexes, SC DNA was detected in the blood between 1 and 60 minutes after injection

(Thierry et al. 1997). OC DNA degrades with a half-life of approximately 10 to 20

minutes. Uptake of pDNA in blood cells reaches a maximum as early as 1 minute after

injection of liposome:pDNA complexes.

A major problem associated with these studies is that the analysis of samples was

done only qualitatively. No attempt was made to quantitate the amounts and types of

plasmid present in the bloodstream at various times. This information is critical for an

evaluation of the predictive value of pharmacokinetic parameters associated with gene

delivery.

Quantitative analysis of gene delivery has been done using IV injected

radiolabeled pDNA, [32p] or [33P], in mice. In these studies, the half-life of naked pDNA

is approximately 10 minutes (Kawabata et al. 1995; Osaka et al. 1996). The total plasma

radioactivity displays a degradation pattern consistent with a two compartment body

model (Mahato et al. 1995). Total body clearance of naked pDNA is estimated at 102

ml/hr, and plasma AUC is estimated at 0.98 (% of dose*hr/ml). Urinary radioactivity

increases with time, indicating the degradation products are excreted via the kidney.

Similar results were obtained following injection of radiolabeled liposome:pDNA








complexes with AUC's of 0.57 to 0.7 (% dose*hr/ml) and total clearance ranging from

175.8 to 142.7 (ml/hr) (Mahato et al. 1995). Half-life for radiolabeled pDNA:

dimethyldioctadecylammonium bromide: dioleoylphosphatidylethanolamine complexes

was shorter ranging from 4 to 8 minutes (Osaka et al. 1996) suggesting rapid tissue

entrapment of the liposome:pDNA complexes relative to naked plasmid. Twenty-four

hours after injection, blood cell and plasma radioactivity for naked pDNA and

liposome:pDNA complexes were similar (Osaka et al. 1996).

Between these 3 analysis methods (agarose gel, Southern blotting, and

radiolabeling) agarose gel analysis can determine more detailed information on the

degradation of different structures of plasmid, (SC, OC, and L). This method is easy to

apply and can be done under normal conditions without the limitations associated with

radioactivity. The disadvantage of this method is that it is traditionally a semi-

quantitative method. The advantage of the [33P] and [32P] methods is that these are

quantitative methods and are more sensitive than agarose gel analysis. The disadvantages

are that radiolabeling yields OC pDNA and thus, this method gives no information on the

pharmacokinetics of SC pDNA. OC plasmid can also not be differentiated from L

pDNA. The radioactivity is also counted without discriminating the degraded DNA

fragments or the free label. Furthermore, special conditions and precautions are needed

to handle radioactive materials. The difference between [32p] and [33p] is that [33P] has

less personal danger and offers greater ease of handling than [32p] (Song et al. 1997;

Niven et al. 1998).

Overall, when pDNA is injected in mice, SC pDNA has not been detected when

administered as naked pDNA, but is after the injection of liposome:pDNA complexes.








After administration as naked pDNA, OC and L pDNA degrades with a half-life of

between 5 and 10 minutes. The half-life of OC or L pDNA after administration of

liposome:pDNA complexes ranges from 4 to 20 minutes. OC pDNA is available for

transcription if taken up by cells. (Adami et al. 1998; Niven et al. 1998) Thus,

administering pDNA in the form of liposome:pDNA complexes may offer a slight

increase in the availability of IV administered pDNA.

Distribution of Plasmid DNA in Tissues after IV In-jection

Tissue distribution of pDNA may be measured using radioactivity, Southern

analysis, or whole body autoradiography. Using Southern analysis, pDNA has been

detected in the bone marrow, heart, kidney, liver, lung, spleen, and muscle as early as I

hour after injection (Lew et al. 1995; Niven et al. 1998). No plasmid was detectable in

the brain, intestine, and ovaries.

Sub-picogram levels may be detected using polymerase chain reaction (PCR).

Using this method, Lew and coworkers showed that at 7 days after IV injection, the range

of residual plasmid was 1 fg/tg in the brain, intestine, and gonads, and was 64 fg/ptg in

the marrow, heart, liver, spleen, and muscle (translating to approximately 250-16,000

copies/genome (Lew et al. 1995). By 28 days post-injection, levels of detectable plasmid

had decreased 128 fold. Using PCR, residual plasmid remained detectable 6 months post

injection at 2 to 8 fg/lag genomic DNA and was predominantly in the muscle.

After injection of radiolabeled plasmid, distribution may be measured by isolating

tissues and measuring homogenates in a scintillation counter (Kawabata et al. 1995;

Mahato et al. 1995). Alternatively, the entire carcass may be measured by whole body

sectioning and autoradiography (Osaka et al. 1996; Niven et al. 1998).








After injection of radiolabeled naked pDNA, accumulation of radioactivity occurs

initially in the lung, but declines rapidly through 1 minute post-injection (Kawabata et al.

1995). Osaka and coworkers found that by 2 minutes after injection of naked pDNA,

organ distribution is liver>spleen>lung, blood (Osaka et al. 1996). Whereas, Niven and

coworkers found the time to reach maximum levels in the lungs is as long as 5 minutes

versus 2 hours in the liver (Niven et al. 1998). Thus, there appears to be an initial rapid

entrapment and transient accumulation in the lungs with accumulation occurring in the

liver after a short period of time. Plasmid DNA was preferentially recovered in the non-

parenchymal cells in the liver suggesting that the liver is acting in a scavenger role in

uptake (Kawabata et al. 1995).

When compared to naked pDNA, IV injection of liposome:pDNA complexes

shows a higher accumulation of radioactivity in the lung 2 minutes after injection, Osaka

and coworkers showed the major organs exhibit a distribution of

lung>liver>spleen>kidney (Osaka et al. 1996). One hour after injection, a slight rise is

seen in most organs, which is probably related to continuous uptake by the

reticuloendothelial system. By 24 hours after injection of liposome:pDNA complexes,

lung radioactivity dropped approximately 70 fold, with a distribution in major organs of

spleen>liver>lung, kidney.

Conclusions

A complete understanding of the classical pharmacokinetic parameters of gene

delivery is necessary to move genetic agents forward as clinical therapeutics. Problems

include the rapid clearance of naked pDNA and liposome:pDNA complexes without

expression of the gene products, poor target tissue specificity, and degradation in the

plasma. After systemic administration in mice, plasmid DNA is rapidly eliminated from





22


the circulation by extensive uptake by the reticuloendothelial system and degradation by

plasma nucleases. Hepatic uptake is almost identical to liver blood flow suggesting

highly efficient uptake. A complete pharmacokinetic model of all 3 forms of plasmid

DNA (SC, OC, and L) has not been proposed. As the products of the biotechnology

industry begin to move towards more clinical applications, the pharmacokinetic modeling

of gene delivery will likely become an intensely investigated area.













CHAPTER 2
PHARMACOKINETICS OF PLASMID DNA IN ISOLATED RAT PLASMA

Introduction

In vivo delivery of plasmid DNA (pDNA) encoding for therapeutic proteins to

patients via parenteral administration is an attractive means by which to target the gene to

a wide variety of tissues. Early studies revealed that endogenous enzymes present in the

plasma play a role in the clearance of nucleotides from the bloodstream (Chused 1972;

Whaley 1972; Chia 1979; Piva 1998). These early studies have displayed that pDNA

incubated in the presence of 10 % fetal bovine serum shows initial degradation by 15 min

and is completely degraded by 60 min (Piva 1998). Similar results have been displayed

in the presence of 90% human serum (Piva 1998).

Nucleases will convert the native supercoiled (SC) pDNA topoform to the open

circular (OC) and linear (L) forms of the plasmid (Lodish 1995). Changes in topoform

have been associated with alterations in transcriptional activity. The significance of this

change has been the matter of some debate. For example, the OC form of the plasmid

has been shown to express similar levels of chloramphenicol acetyl transferase and

luciferase proteins (Adami et al. 1998; Niven et al. 1998) to 2 to 4 times less (Hirose

1993; Cherng 1999; Ramsey 1999) levels of transcribed luciferase and lac-Z, proteins.

Increases in the amount of supercoiling serves to further increase the percent maximal

transcription (Ramsey 1999). Furthermore, the time required for formation of the

transcription preinitiation complex has been shown to be decreased with a SC template

(Hirose 1993). Degradation to the L form of pDNA is associated with significant losses








in transcriptional activity (90-100%) (Hirose 1993; Adami et al. 1998; Niven et al. 1998;

Cherng 1999; Ramsey 1999). Differences in transcriptional activity may need to be

accounted for in future pharamcodynamic studies.

Early studies revealed that serum nucleases play a role in the rapid clearance of

genomic DNA from the circulation of injected animals (Gosse et al. 1965). Recent

studies on the pharmacokinetics of pDNA have attempted to use radiolabeled pDNA for

detection (Osaka et al. 1996; Niven et al. 1998). However, the radiolabeling procedure

involves nick translation, thereby eliminating the possibility of maintaining the SC

topoform. Furthermore, this method does not discriminate the degraded pDNA from the

intact plasmid, thus yielding an overestimation of the true half-life of the intact pDNA.

Other studies on the pharmacokinetics of SC and OC pDNA have been only qualitative

citing the presence of pDNA topoforms at various time points (Kawabata et al. 1995;

Osaka et al. 1996; Thierry et al. 1997).

Thierry and coworkers studied the stability of pDNA in the bloodstream of mice

after IV injection (Thierry et al. 1997). Their results indicated that SC plasmid was not

detectable in the plasma or red blood cell fractions 1 min after injection of pDNA. The

true half-life was unable to be calculated using their method due to this rapid degradation.

Kawabata and coworkers found that the SC pDNA was completely converted to the OC

topoform within 5 min when incubated in mouse whole blood (Kawabata et al. 1995).

Little other information on the pharmacokinetics of pDNA is available. The exact

pharmacokinetics underlying this rapid degradative process is not fully understood. To

properly dose and reach the desired therapeutic endpoints, a thorough understanding of

the pharmacokinetics of pDNA is a necessity.








It is necessary to study the effects of plasma on pDNA in order to begin

understanding the importance of the degradation of pDNA in the blood and allow a

foundation upon which comparisons of delivery vehicles can be made. Naked pDNA has

been shown to remain in the plasma fraction of blood (Osaka et al. 1996). For these

reasons, we sought to investigate the pharmacokinetic processes underlying the stability

of pDNA in a rat plasma model. We further sought to construct a complete

pharmacokinetic model to describe the degradation of all three topoforms of pDNA in

plasma. This model will allow a prediction of the time course of potential tissue

exposure to the transcriptionally active SC and OC pDNA topoforms.

Methods

Phenol: chloroform: isoamyl alcohol (25: 24: 1, v/v/v), Tris, boric acid, EDTA,

and agarose were purchased from Sigma Chemical Company (St. Louis, MO). Ethidium

bromide (electrophoresis grade) was purchased from Fisher Biotech (Fair Lawn, NJ).

Competent JM 109 bacteria (Promega, Madison, WI) were transformed according to the

manufacturers directions with the pGL3 control plasmid (Promega, Madison, WI),

pGeneMax-Luciferase (Gene Therapy Systems, San Francisco, CA) or pGE 150 plasmid

(a generous gift of Dr. G. Elliot, Marie Curie Research Institute, The Chart, Oxted,

Surrey, UK). Representative plasmid maps are presented in Figures 2-1, 2-2, and 2-3 for

the pGL3, pGeneMax-Luciferase, and pGE150 plasmids, respectively. Plasmid DNA

was isolated from overnight cultures using the Plasmid Maxi-Prep kit (Quiagen,

Valencia, CA), and was >95% SC by agarose gel analysis.

Blood was isolated from male Sprague-Dawley rats (300-350 g) by cardiac

puncture, and immediately placed in heparinized test tubes (Vacutainer, Becton
















































Figure 2-1. Plasmid map of pGL3 Control.












































Figure 2-2. Plasmid map of the pGeneMax-Luciferase.














































Figure 2-3. Plasmid map ofpGE150.








Dickinson, Franklin Lakes, NJ) on ice at the times indicated. Blood samples were

centrifuged at 6,000 g for 5 min. For dilution experiments, plasma was diluted to 25 and

50% with PBS or PBS containing 0.1 mM EDTA. To analyze the effects of heat, plasma

samples were incubated at 90'C for 10 min in sealed tubes before assay. Plasma (600 p)

was removed and placed on ice until assay. Plasma samples were warmed to 370 C in a

water bath and maintained at 370 C for the duration of the experiment. Plasmid DNA (12

tl/17 jtg) in TE buffer was incubated in the 370 C plasma and 50 p.1 samples were taken

at various times. Phenol: chloroform: isoamyl alcohol (25: 24: 1, v/v/v) (80 p1) was

immediately added to each sample, vortexed for 5 s at low speed, and placed on ice.

Samples were centrifuged at 20,800 x g for 10 min at room temperature. From the

supernatant, an aliquot of 15 p.1 was removed, 5 p.l of 6 x loading dye (Promega,

Madison, WI) added and placed on ice until loaded on an agarose gel.

Samples were loaded on 0.8% agarose in 0.9 M Tris-Borate and 1 mM EDTA

gels containing 0.001% ethidium bromide. Electrophoresis was carried out at 0.30 V/cm3

for 12 h. The ethidium bromide was excited on a UV light box (Model TFX-35M, Life

Technologies, Grand Island, NY) and the net fluorescence intensity captured at 590 nm

on a Kodak DC120 digital camera (Eastman Kodak, Rochester, NY). The amounts of

SC, OC, and L pDNA were calculated using Kodak Digital Science 1D Image Analysis

Software (version 3.0, Eastman Kodak Company, Rochester NY) with a Lambda Hind III

digest (Promega, Madison WI) and a High DNA Mass Ladder (Life Technologies, Grand

Island, NY) as external standards. Accuracy was 96 to 104% for SC pDNA, 98 to 108%

for OC, and 96 to 113% for L pDNA. Percent coefficient of variation was < 5%, 19%,

and 13% for SC, OC and L forms of the plasmid respectively. Standard curves for all








three forms of the plasmid (Figures 2-4, 2-5, and 2-6) were linear between 10 and 250 ng

pDNA bands (R2 = 0.9995, 0.9985, and 0.9933 for SC, OC, and L respectively). All

reported concentrations were calculated from bands within the range of the standard

curves. Lower limit of quantitation was 0.5 ng4. for all three forms of the plasmid.

Lower limit of detection was 0.25 ng/tl for all three forms of the plasmid. Method

parameters are summarized in Table 2-1. Comparisons of the relative fluorescence of SC

pDNA versus OC and L pDNA were made by analyzing equivalent amounts as described

above and comparing the resulting fluorescence. It was found that on a weight to weight

ratio, SC pDNA was only 59% as fluorescent relative to L pDNA by agarose gel analysis.

To correct for this difference, SC pDNA amounts were multiplied by 1.7 prior to

analysis. This difference has been reported previously and is likely due to the relative

inaccessibility of ethidium bromide to the SC topology (Cantor 1980). Percent recovery

using the phenol: chloroform: isoamyl alcohol method was found not to be dependent on

topoform. Recovery was 90 ( 6) % for SC and 86 ( 13) % for L pDNA. Comparisons

of the relative fluorescence of SC pDNA versus L pDNA were made by digesting SC

pDNA with the Hind III restriction enzyme (Promega, Madison, WI) which has a single

recognition site in the plasmid. Equivalent amounts of L and SC pDNA were then loaded

on agarose gels as described above and the relative fluorescence compared. Percent

recovery was calculated by comparing phenol: chloroform: isoamyl alcohol (25: 24: 1,

v/v/v) extracted versus non-extracted known amounts and analyzing on agarose gels as

described above.

















300


250


200



JIOO

100-


50


0 fI I
0 50 100 150 200 250
A260 DNA equivalents



Figure 2-4. Standard curve for supercoiled pDNA. Abcissa represents total ng estimated
by UV absorbance at 260 nm. Ordinate represents ng calculated by the KDS ID
software. Error bars represent 1 standard deviation.





















300


250-
R2 =0.9985

200


150
U




50



0-
0 50 100 150 200 250
A260 DNA Equivalents






Figure 2-5. Standard curve for OC pDNA. Abcissa represents total ng estimated by UV
absorbance at 260 nm. Ordinate represents ng calculated by the KDS ID software. Error
bars represent 1 standard deviation.

















300



250



200


150


R2 = 0.9933


100



50


0
0.I I I I I
0 50 100 150 200 250
A260 DNA equivalents




Figure 2-6. Standard curve for Linear pDNA. Abcissa represents total ng estimated by
UV absorbance at 260 nm. Ordinate represents ng calculated by the KDS ID software.
Error bars represent 1 standard deviation.


















Table 2-1. Method parameters for pDNA analysis.


Accuracy Supercoiled: 94-101 %
Open Circular: 98-108 %
Linear: 96-113 %
Precision Supercoled: 5 %
Open Cicular: 19 %
Linear: 13 %
Lower Limit of Quantitation 0.5 ng/pt
Lower Limit of Detection 0.25 ng/gl
Recovery from Plasma Supercoiled: 90 (6) %
Linear: 86 (13) %










Theoretical

The degradation of SC pDNA was assumed to follow pseudo first-order kinetics.

The model used is diagrammed in Figure 2-7. In this model, pDNA degradation is

considered to be a unidirectional process. The degradation of L pDNA is considered to

yield fragments of heterogeneous lengths, thus these products were not included in the

fitted model. No elimination from any of the compartments is assumed to occur through

routes other than degradation to the following topoform.

Based on this model the following differential equations were derived to describe

the process:


dSC k *SC
dt
dOC
-ks SC kOC
dt
= k OC- k, L
dt
The amounts of supercoiled, open circular and linear pDNA were then fit to the

integrated form of the equations:


SC = SCo e- .
o c = k,. SC o .( ,', .e -k.-, + I e-k ,t
Lo= k, SCo C(,.( 1 k.., + I .e-k, t +e-kt
-( k o(k,- ko e +ko ) (k,-kXk,-k,) (k.-kXk,-k, )
Where SC, OC, and L are the amounts of supercoiled, open circular, and linear pDNA

present at time--t, respectively. SCo is the amount of supercoiled pDNA present at time

(t)=O. The constants k, ,k,, and k, represent the rate constants for the degradation of SC,

OC, and L pDNA respectively. The constants represent the activity of all enzymes acting

in the degradation process. Non-linear curve fitting and goodness of fit, model selection








criteria (MSC) assessment was carried out using Scientist (version 4.0, Micromath, Salt

Lake City, UT) (MicroMath 1995). Area under the plasma concentration time curve

(AUC) was calculated using trapezoidal rule. Area under the terminal portion of the

plasma concentration time curve, AUCterm, was calculated by integration using the

equation:


AUT C -cast
AU'term- k
Where Cast is the last concentration point measured and k is the terminal elimination rate

constant. Clearance (Cl) was calculated from the volume (V) of rat plasma (7.8 ml) and

the terminal elimination rate constant (k) using the equation (Davies 1993):



Cl- V-k


Statistical analysis was performed using SAS (The SAS Institute, Cary, NC).

Results

For quantitative purposes, the relative fluorescence of SC pDNA was compared to

that of OC and L pDNA. It was found that on a weight to weight ratio, SC pDNA was

only 59% as fluorescent relative to L pDNA by agarose gel analysis. To correct for this

difference, SC pDNA amounts were multiplied by 1.7 prior to analysis. This difference

has been reported previously, and is likely due to the relative inaccessibility of ethidium

bromide to the SC topology (Cantor 1980). Percent recovery using the phenol:

chloroform: isoamyl alcohol method was found not to be dependent on topoform.

Recovery was 90 (6) % for SC and 86 (13) % for L pDNA.





















is iks [ko
sc oc OC___ > Linear



Figure 2-7. Pharmacokinetic model of plasmid DNA degradation in rat plasma. The
model is considered to be a unidirectional process. SC, OC, and L represent the amounts
of supercoiled, open circular, and linear plasmid, respectively, in each compartment. The
rate constants ks, k,, and k, represent the degradation constants for supercoiled, open
circular, and linear plasmid, respectively.










Figure 2-8 displays a representative gel in which the degradation of SC pDNA

and the appearance of OC and L topoforms of plasmid is observed. In addition, the

degradation products of L pDNA are visible as a light smear running below the band at

60 min. Under the conditions used in this experiment, limit of quantification was 0.5

ng/ d using the Lambda Hind III size standard. Plasmid amounts were calculated from

agarose gel analysis using Kodak Digital Science 1D image analysis software (Eastman

Kodak, Rochester, NY) as described in the methods section. The observed and predicted

values, based on the model displayed in Figure 2-8, are plotted in Figure 2-9. Plasmid

concentrations were well described the model, MSC=3.0. Pharmacokinetic parameters

calculated based on the model are summarized in Table 2-2. SC pDNA degraded rapidly

in the plasma with a half-life of 1.2 ( 0.1) min. OC plasmid however was fairly stable,

degrading with a half-life of 21 ( 1) min. L plasmid degraded more rapidly than the OC

topoform but was fairly stable, in comparison to the SC plasmid degrading with a half-

life of 11 ( 2) min. OC AUC was nearly 17 times larger than SC, and 2.3 times larger

than L pDNA (Table 2-3).

No kinetics suggestive of enzyme saturation were observed under the

experimental conditions tested. However, to ensure that saturation of plasma nucleases

was not resulting in artificially low rate constant values, we analyzed the rate constants

produced in dilute plasma (dilution was chosen as decreasing the dose of pDNA quickly

results in a loss of sensitivity and sample sizes too large for loading). If saturation of


















1 2 3 4 5 6 7 8 9 10 11 12


Oc

Linear


Figure 2-8. Agarose gel analysis of pDNA degradation in isolated rat plasma. Lane 1;
size standard, lane 2; 30 sec, lane 3; 1 min, lane 4; 2 min, lane 5; 3 min, lane 6; 5 min,
lane 7; 10 min, lane 8; 20 min, lane 9; 30 min, lane 10; 45 min, lane 11; 60 min, lane 12;
80 min.






40

















15.0- I


12.5-


10.0

tM 7.5 --
5.o5
z
0. 5.0-


2.5-


0.0-
0 10 20 30 40 50 60

time (min)
Figure 2-9. Experimental and fitted data based on the pharmacokinetic model described
in the text. Data points represent actual experimental data. Lines represent values
predicted by the model. Data represents mean of n=3 1 standard deviation. Key: U
supercoiled, open circular, A linear.

















Table 2-2. Pharmacokinetic parameters for pDNA in isolated rat plasma


Topoform Rate Value (min') Standard Half-life (min)
constant Deviation
Supercoiled k, 0.6 0.03 1.2 0.1)

Open circular k, 0.03 0.002 21 (+ 1)

Linear k, 0.06 0.008 11 (2)

Data represent the fitted values of n=6 1 standard deviation.


















Table 2-3. Noncompartmental parameters for pDNA after incubation in isolated plasma.


Topoform AUC (ng/ l*min) Clearance
( I/min)
Supercoiled 18 360 (+ 9)
Open circular 310 23 (+ 1)
Linear 130 47 (5)

Data represent n=6 standard deviation. AUC was calculated from the model fitted
values using trapezoidal rule as described in the methods section. Clearance was
calculated from the fitted rate constants and volume of rat plasma as described in the
Methods section.










plasma nucleases was occurring, we expected that the rate constants in dilute plasma

should deviate from a linear relationship negatively. Thus we tested the kinetics of

pDNA degradation in 25% and 50% plasma. As displayed in Figure 2-10, no deviation

was observed in the degradation of SC and OC pDNA.

To further investigate the mechanism responsible for the observed degradation,

and further validate our assay (to ensure the assay was not causing degradation itself) we

studied the degradation of pDNA in PBS diluted 25% heated plasma (90'C for 10 min)

and PBS containing 0.1 mM EDTA. No degradation of SC pDNA was observed in either

case through 1 hr (Figure 2-11). The degradation sensitivity to heat and EDTA provides

evidence that the degradation observed in the assay is due to enzymatic processes.

We next sought to determine if the degradation observed in the previous

experiments was dependent upon pDNA sequence. We, therefore, utilized the same

model diagrammed above and replaced the pGL3 plasmid with the pGE150 plasmid.

Unlike the pGL3 plasmid, which encodes for the luciferase protein and has an SV40

promoter, this plasmid encodes for the green fluorescent protein and includes a CMV

promoter. If the degradation of pDNA in the plasma was sequence dependent, we

expected the degradation rate constants observed to differ from those observed in the

previous experiment. A comparison of plasma concentrations of OC and L pDNA is

presented in Figures 2-12 and 2-13. The resulting rate constants are presented in Table 4.

Again the model diagrammed in Figure 2-7 described the data (model selection

criteria = 3.1, R2=0.96, 0.99, and 0.92 for SC, OC, and L respectively). To determine if




















y = 0.6494x- 0.0311
R2 = 0.9361 .o


y = 0.0415x 0.0082
R2 = 0.9962


*Ks
=Ko






1.2


% plasma


Figure 2-10. Analysis of rate constants in dilute plasma. Degradation rate constants were
modeled in PBS diluted rat plasma. Rate constants represent the fitted values of n=6 rats/
time point. Key: *ks in dilute plasma, k, in dilute plasma. The value of k, is not
reported due to the prolonged stability of linear plasmid in dilute plasma.




















A B












Std O.5m lm 2m3m 5m l0m 15m 20m 30m45m lhr Std O.5m Im2m 5m 1Om 20m 30m45m Ilhr

Figure 2-11. Degradation of supercoiled pDNA in 25% rat plasma after (A) incubating
the plasma at 90'C for 10 min (B) the addition of 0.1 mM EDTA.








these rates were significantly different from those obtained using the pGL3 plasmid a

statistical analysis was carried out using a 2-tailed equal variance student's t-test. The

resulting parameters (ko, and k1) were not significantly different when judged at the

p<0.05 criteria. These results suggest that pDNA sequence is not a major factor involved

in the overall degradation of pDNA by plasma nucleases.
Conclusions

Previous reports on the pharmacokinetics of pDNA have only been qualitative, or

involved radiolabeling. These studies indicated that pDNA degrades within 5 minutes in

vitro or after IV injection (Kawabata et al. 1995; Thierry et al. 1997). In this study, we

sought to quantitatively model the pharmacokinetics underlying the stability of pDNA in

the plasma. The results revealed that SC pDNA degrades in the plasma with a half-life of

1 min. OC pDNA is more stable than the SC topoform degrading with a half-life of 20

min. L pDNA is degraded more rapidly than the OC topoform. This latter shortened

stability is likely due to the accessibility of various nucleases present in the plasma to the

L pDNA. OC plasmid must be nicked by endonucleases on each sister strand in the same

location to generate L pDNA. However L pDNA would be accessible to both

endonucleases and exonucleases, thus degrading more rapidly. The model and equations

presented successfully described the degradation of pDNA in the plasma.

Investigations by Thierry and coworkers suggested the main nuclease activity was

endonucleolytic based on the finding that the L: SC ratio increased over time and the SC:

OC ratio remained identical to control (Thierry et al. 1997). However this view fails to
















100

10

1

0.1


20


40


60


80


time (min)




Figure 2-12. Comparison of concentrations of OC pDNA using (*) pGE150
concentrations of OC pDNA using (U) pGL3 in isolated rat plasma. Data represents
mean of n=3 1 standard deviation.

















10



zO0.1
0.01

0 20 40 60 80

time (min)

Figure 2-13. Comparison of concentrations of L pDNA using (4) pGE150 versus
concentrations of L pDNA using (0) pGL3 in isolated rat plasma. Data represents mean
of n=3 1 standard deviation.




















Table 2-4. Pharmacokinetic parameters for pDNA after incubating the pGE150 plasmid
in isolated rat plasma.

Standard

Topoform Rate constant Value (min-1) Deviation Half-life (min)

Open Circular k, 0.04 0.007 21 (1)

Linear k, 0.06 0.007 11 (1)

Data represent the fitted values of n=6 rats.








recognize endonucleolytic activity on L pDNA also generates degradation products. If

endouclease activity is the primary route of degradation, the kinetic ratios should all

remain similar or decrease, owing to the fact that both exonucleases and endonucleases

are active on the L pDNA and are thus both responsible for the observed degradation.

Our results suggest that L pDNA has faster kinetics. This can be explained by

endonuclease activity generating more free ends for degradation by exonucleases,

exonucleases are more active than endonucleases, or that topoform influences the binding

of these enzymes and thus influences reaction rate. Thus the main nuclease activity

responsible for the observed kinetics remains to be answered.

Area under the curve analysis revealed that tissues would be exposed to the OC

topoform predominantly after injection of naked pDNA (Table 2-2). Blood flow through

any individual organ becomes the limiting factor in its ability to uptake a drug, which is

highly metabolized in the plasma. When compared to hepatic plasma flow in the rat

(8.14 ml/min), clearance values for the degradation of SC plasmid (4.6 ml/min) suggest

that metabolism in the bloodstream is a major pathway by which in vivo clearance of SC

pDNA can occur (Davies 1993). However, given that the clearance by degradation in the

plasma is less than the liver blood flow, it also suggests that the liver possess a perfusion

rate sufficient for uptake of SC pDNA after IV injection. This parallels the findings of

Kawabata and coworkers who observed that naked plasmid was cleared more rapidly

from the circulation after IV injection than after in vitro incubation in whole blood

(Kawabata et al. 1995). Lung, kidney, and spleen have also been shown to take up

detectable amounts of plasmid after IV injection (Kawabata et al. 1995; Osaka et al.








1996). Our model establishes not only that tissue uptake of plasmid is possible, but also

that tissue uptake of the non-nicked SC topoform is possible after IV injection.

In summary this study presents a pharmacokinetic model describing the

degradation of pDNA in rat plasma. A pharmacokinetic model is presented that can be of

use in the future as gene therapy moves toward clinical trials. Using the derived model,

we are able to conclude that naked SC pDNA degrades in rat plasma with a half-life of

1.2 ( 0.05) min, OC with a half-life of 21 ( 1) min, and L pDNA with a half-life of 11

( 2) min.













CHAPTER 3
PHARMACOKINETICS OF PLASMID DNA AFTER IV BOLUS ADMINISTRATION
IN THE RAT

Introduction

Naked pDNA is being used successfully in gene delivery by administration IM or

SQ, (Haensler et al. 1999; Noll et al. 1999; Osorio et al. 1999; Rizzuto et al. 1999) and

after IV injection (Wang et al. 1995; Budker et al. 1998; Song et al. 1998; Liu 1999;

Zhang et al. 1999) in rats and mice. The success in these studies indicates that gene

therapy is an attractive means by which to achieve therapeutic response. Thus, a

thorough understanding of the pharmacokinetics of naked pDNA is an important area to

be considered in order to move towards use in clinical trials

The pharmacokinetics of pDNA after IV bolus administration have been

investigated using radiolabeling with linearized [33p] pDNA (Osaka et al. 1996). These

investigations have led to the conclusion that the half-life of the pDNA radiolabel is 7 to

12 min after IV bolus administration of naked pDNA in mice. However, this analysis

offers no information on the other functional forms of the plasmid; supercoiled (SC),

open circular (OC), or linear (L), nor does it discriminate the free label. Other studies

have qualitatively revealed that the SC topoform of pDNA is not detectable as early as

one minute post IV injection in mice (Lew et al. 1995; Thierry et al. 1997). The OC form

of the plasmid has a half-life estimated in these studies to be in the range of 10 to 20

minutes (Thierry et al. 1997).








The purpose of this investigation was to model the pharmacokinetics of naked

pDNA in a topoform specific manner after IV bolus administration in the rat. We further

sought to determine if the observed pharmacokinetics were affected by changes in

plasmid sequence. These results were then compared to pDNA degradation in isolated

plasma in order to determine the relative importance of plasma nucleases in the

pharmacokinetics of pDNA.

Methods

Animals (male Sprague-Dawley rats 300-350 g) were purchased from Charles

River Laboratories (Wilmington, MA). Animals were housed in the University of Florida

Animal Resources Unit prior to all experiments and were given food and water ad

libitum. Animals were anesthetized by intraperitoneal injection with 0.5 ml of a cocktail

containing 13 mg/kg Xylazine, 2.15 mg/kg Acepromazine (The Butler Co., Columbus,

OH), and 66 mg/kg Ketamine (Fort Dodge Animal Health, Fort Dodge Iowa).

Phenol: chloroform: isoamyl alcohol (25: 24: 1, v/v/v), chloroform, Tris, boric

acid, EDTA, and agarose were purchased from Sigma Chemical Company (St. Louis,

MO). Ethidium bromide (electrophoresis grade) was purchased from Fisher Biotech

(Fair Lawn, NJ). Competent JM109 bacteria (Promega, Madison, WI) were transformed

according to the manufacturer's directions with the pGL3 control plasmid (Promega,

Madison, WI) or pGeneMax plasmid (Gene Therapy Systems, San Francisco, CA).

Plasmid DNA was isolated from overnight cultures using alkaline lysis and

ultracentrifugation with a CsCl gradient (Katz 1977). Isolated pDNA was resuspended in

phosphate buffered saline. Plasmid was >90% SC by agarose gel analysis.

To facilitate blood sampling, male Sprague-Dawley rats (300-350g) were

anesthetized and the jugular vein was exposed via an incision, isolated, ligated, and








nicked with ophthalmic scissors. A sterile silatstic (0.640 cm internal diameter by 0.12

cm outer diameter, 10 cm in length) filled with sterile saline was threaded 3 0-40 mm into

the jugular vein and positioned just distal to the entrance to the right atrium and secured

by 6.0 silk sutures Figure 3-1). For injections, the femoral vein was isolated, and pDNA

was injected into the femoral vein using a 27-gauge needle (Figure 3-2). Isolated blood

samples (approx. 300 tl) were drawn through the jugular vein cannula and immediately

placed in test tubes containing 0.57 ml of 0.34 M EDTA (Vacutainer, Becton Dickinson,

Franklin Lakes, NJ) on ice at the times indicated. This concentration of EDTA has

previously been shown to inhibit the degradation of pDNA in isolated rat plasma (Houk

1999).

To isolate pDNA from whole blood samples, 250 [d1 of blood was liquid/ liquid

extracted with 250 [d1 of phenol: chloroform: isoamyl alcohol (25: 24: 1, v/v/v), vortexed

for 5 s at low speed, and centrifuged at 20,800 x g for 10 min at room temperature. The

aqueous phase was removed and stored at -20'C until analysis.

Samples were loaded on 0.8% agarose in 0.9 M Tris-borate, 1 mM EDTA gels

containing 0.001% ethidium bromide. Electrophoresis was carried out at 0.30 V/cm3 for

12h. The ethidium bromide was excited on a UV light box (Model TFX-35M, Life

Technologies, Grand Island, NY) and the net fluorescence intensity captured at 590 nm

on a Kodak DC120 digital camera (Eastman Kodak, Rochester, NY). The amounts of

SC, OC, and L pDNA were calculated using Kodak Digital Science 1D Image Analysis

Software (version 3.0, Eastman Kodak Company, Rochester NY) with a Lambda Hind III

digest (Promega, Madison WI) and a High DNA Mass Ladder (Life Technologies, Grand











































Figure 3-1. Photograph of the jugular cannula placement used for blood sampling.












































Figure 3-2. Photograph of the femoral vein isolation and injection procedure used for IV
bolus administration.










Island, NY) as external standards. Accuracy was 96 to 104% for SC pDNA, 98 to 108%

for OC, and 96 to 113% for L pDNA. Percent coefficient of variation was < 5%, 19%,

and 13% for SC, OC and L forms of the plasmid respectively. Standard curves for all

three forms of the plasmid (Figures 2-1, 2-2, and 2-3) were linear between 10 and 250 ng

pDNA bands (R2 = 0.9995, 0.9985, and 0.9933 for SC, OC, and L, respectively). All

reported concentrations were calculated from bands within the range of the standard

curves. Lower limit of quantitation was 0.5 ng/[tl for all three forms of the plasmid.

Lower limit of detection was 0.25 ng/pll for all three forms of the plasmid. Method

parameters are summarized in Table 2-1. Comparisons of the relative fluorescence of SC

pDNA versus OC and L pDNA were made by analyzing equivalent amounts as described

above and comparing the resulting fluorescence. It was found that on a weight to weight

ratio, SC pDNA was only 59% as fluorescent relative to L pDNA by agarose gel analysis.

To correct for this difference, SC pDNA amounts were multiplied by 1.7 prior to

analysis. This difference has been reported previously, and is likely due to the relative

inaccessibility of ethidium bromide to the SC topology (Cantor 1980). Percent recovery

using the phenol: chloroform: isoamyl alcohol method was found not to be dependent on

topoform. Recovery was 90 ( 6) % for SC and 86 ( 13) % for L pDNA.

Theoretica

The degradation of SC pDNA was assumed to follow pseudo first-order kinetics.

The model used is diagrammed in Figure 2-3. In this model, pDNA degradation is

considered to be a unidirectional process. The degradation of L pDNA is considered to

yield fragments of heterogeneous lengths, thus these products were not included in the







fitted model. No elimination from any of the compartments is assumed to occur through

routes other than degradation to the following topoform.

Based on this model the following differential equations were derived to describe

the process:


dSC k SC
dt
dOC~k
dt= kS.SC-ko.OC
dt
dLk0
A- = ko.OC- k, .L
dt



The amounts of supercoiled, open circular and linear pDNA were then fit to the

integrated form of the equations:


SC = SC0 e-k,t
OC = ks SCo ( kIk, e kot + k,--k, .ek t )
-ko ks o ( kk e e + e -kt)
(k,-ko)(k,-ko) + (ko -k, )(k, -k,) "e + (ko kj)(k, kj)e
Where SC, OC, and L are the amounts of supercoiled, open circular, and linear

pDNA present at time=t, respectively. SC0 is the amount of supercoiled pDNA present at

time (t)=O. The constants ks ,ko, and k, represent the rate constants for the degradation of

supercoiled, open circular, and linear pDNA, respectively. The constants represent the

activity of all enzymes acting in the degradation process. Non-linear curve fitting and

statistical analysis was carried out using Scientist (version 4.0, Micromath, Salt Lake



















1 2 3 4 5 6 7 8 9 10


Figure 3-3. A representative gel from which plasmid amounts were quantified as
described in the methods section. Lane 1: size standard, lane 2: 1 min, lane 3: 2 min, lane
4: 3.5 min, lane 5: 5 min, lane 6:10 min, lane 7: 20 min, lane 8: 30 min, lane 9: 45 min,
lane 10: 60 min.








City, UT). Noncompartmental pharmacokinetic analysis was carried out using standard

parameters (Gibaldi 1982).

Result

SC pDNA was not detected as early as 30 seconds post-injection. The OC and L

forms of the pDNA remained detectable through 30 minutes post-injection of the 500 tg

dose. An agarose gel analysis of the isolated samples is presented in Figure 3-3.

An important parameter to be considered is the initial concentrations achieved

after IV administration in comparison to the initial concentrations in vitro. The initial

concentrations of SC pDNA in the in vitro experiments (i.e. the time=0 concentration)

were 10 ( 0.3) ng/ll. After IV bolus administration, the initial extrapolated SC pDNA

concentrations were 17 ( 5) ng/jul. Thus, we concluded that these concentrations were

within a range relevant for comparison. The observed and fitted concentrations of OC

and L pDNA are presented in Figure 3-4. The model again adequately described the

data, model selection criteria=4.42. Pharmacokinetic parameters calculated based upon

the model are presented in Table 3-1.

A comparison of the in vitro and in vivo concentrations of OC and L pDNA are

presented in Figures 3-5 and 3-6, respectively. Calculated pharmacokinetic parameters

are presented in Table 3-2. OC pDNA half-life was markedly shorter after IV bolus

administration than after incubation in isolated plasma, 5.3 (1.4) versus 21 (1) min. L

pDNA removal was also more rapid after IV bolus administration, 1.9 (0.8) versus 11

(2) min after incubation in isolated plasma.

In order to further investigate the importance of plasmid sequence on the observed

pharmacokinetics, we injected the pGeneMax-Luciferase, and pGE150 plasmids by IV








bolus administration at equivalent dose (500 jig). Concentrations of OC and L pDNA in

the bloodstream are presented in Figures 3-7 and 3-8 respectively. The fitted elimination

rate constants for OC and L pDNA were compared by 2-way ANOVA. The results are

displayed in Table 3-3. There were no significant differences between the terminal rate

constants of any of the 3 plasmids by 2-way ANOVA when judged at the p<0.05 criteria.

Conclusions

DNase I is a well characterized enzyme in human plasma present at

concentrations averaging 26.1 (9.2) ng/ml in the sera of normal humans (Chitrabamrung

1981). Traditionally, the presence of this enzyme has led to the conclusion that pDNA

administered IV is degraded rapidly (Gosse et al. 1965; Chused 1972). This has led to

the current view of gene delivery, where protection from plasma nucleases is a major

goal of delivery systems. The results of this study demonstrate that although the half-life

of SC and OC pDNA is remarkably short, degradation alone was not enough to explain

the rapid disappearance of pDNA from the circulation observed in vivo. After IV bolus

the rate of degradation of SC pDNA was greater than 7 times faster than in isolated

plasma (Houk 1999).

Chused and coworkers (Chused 1972) also suggested that nuclease activity was

not enough to explain the rapid clearance of KB cell DNA from the circulation in mice.

In this study, only 2 to 3 % of the radioactivity was hydrolyzed to trichloroacetic acid

(TCA) soluble fragments in 30 min, which was several half-lives longer than in the

circulation. Tsumita and Iwanga (Tsumita and Iwanga 1963) also found that less than 5

% of the total radioactivity was found in the TCA soluble fraction after 4.5 hours in

mouse serum.





62













20-



15 +



10
z



5



0-
0 10 20 30 40 50
time (min)

Figure 3-4. Experimental and fitted data based on the pharmacokinetic model described
in the text. Data points represent actual experimental data. Lines represent values
predicted by the model. Data represents mean of n=6 1 standard devaition. Key: 0
open circular, A linear.
















20


20 40 60


time (min)




Figure 3-5. Concentrations of OC pGL3 after 0: IV bolus administration of a 500 [tg
dose of SC pGL3, and *: Incubation of SC pGL3 in isolated plasma at 37'C.




64









5

3

<2
z
CL

0 20 40 60 80
time (min)

Figure 3-6. Concentrations of L pGL3 after U: IV bolus administration of a 500 [tg dose
of SC pGL3, and *: Incubation of SC pGL3 in isolated plasma at 37*C.



















Table 3-1. Pharmacokinetic parameters calculated after 500 ptg dose of SC pDNA.

Topoform Rate Value Standard Half-life

constant (min-1) Deviation (min)
Supercoiled k, 3.4 0.4 0.2 ( 0.03)
Open circular ko 0.14 0.04 5.3(+ 1.4)
Linear k 0.41 0.18 1.9(+ 0.8)
Parameters represent averages of n=6 rats.




















Table 3-2. Comparison of in vivo and in vitro pharmacokinetic parameters for pDNA.


Topoform Terminal Half-life AUCco (ng/Jil*min) Cl/f (gl/min)
(min)

In vitro In vivo In vitro In vivo In vitro In vivo

Supercoiled 1.2 (0.1) ? 17( 5) N/A 360 ( 9) N/A




Open Circular 21 ( 1) 5.3 (1.4) 280 128 ( 52) 23 (+ 1) 4800
(150) (2000)



Linear 11( 2) 1.9 (0.8) 103 ( 47) 49 ( 28) 47 (5) 11000
(5000)


Parameters represent averages of n=3 (I 1 standard deviation).















100

pCMV-Luc:i
0)
pGE150
,< pGL3
z 1
a

0. 1
0 5 10 15
time (min)
Figure 3-7. Concentrations of OC pDNA in the bloodstream after IV bolus
administration of 500 Lg of pCMV-Luc, pGE150, or pGL3. Data represents mean of n=3
1 standard deviation.
















pCMV-Luc
-IS- pGEI 50
pGL3


0.01


time (min)


Figure 3-8. Concentrations of L pDNA in the bloodstream after IV bolus administration
of 500 [tg of pCMV-Luc, pGE150, or pGL3. Data represents mean of n=3 1 standard
deviation.


1

0.1




















Table 3-3. Comparison of OC and L pDNA parameters after administration of SC pGL3,
pGE150, and pGeneMax.


Plasmid Parameter OC Value L Value
pGL3 AUCJ. 120 (50) 52 (25)
(ng/jtl*min)
Cmax (ng/gl) 13 (4) 3.2 (1.0)
pGE150 AUC.C 160 (30) 55 (12)
(ng/!tl*min)
Cmx (ng/ d) 14 (3) 3.3 (0.9)
pGeneMax AUC.O 121 (25) 59 (3)
(ng/gl*min)
C., (ng/.l) 12 (5) 3.7 (0.3)
Parameters represent averages of n=3 (_ 1 standard deviation).








Alternatively, Gosse and coworkers suggested a major role for nucleases in the

initial degradation of DNA after IV administration in rabbits and mice (Gosse et al.

1965). This finding was based upon the proportionality between the initial rate of

depolymerization and the plasma DNase activity level. Also, a rapid decrease in

viscosity of isolated blood was discovered indicating a depolymerization of DNA.

Finally, a markedly slower disappearance of DNA-methyl green complex (a non-specific

DNase inhibitor) than after native DNA.

The reason for this disparity in results deserves further investigation. Gosse

utilized much higher doses of pDNA than Chused and coworkers in their investigations,

200 p.g versus 5 jtg pDNA in Chused and coworkers 's investigations. This disparity

may be due to saturation of a scavenger receptor, allowing nuclease activity to become

increasingly important. The effect of increasing dose on the clearance of DNA deserves

further investigation.

The results presented in the present study indicate that SC pDNA was

undetectable after IV bolus administration, whereas SC pDNA was readily detectable in

isolated plasma, and remained detectable through 3 min of incubation. Similar results

were seen for the OC and L forms of the plasmid. The half-lives of OC and L pDNA

decreased from 21 (1) to 5.3 (1.4) and 11 (2) to 1.9 (0.8) min, respectively. Thus

indicating that nuclease activity alone is not sufficient to describe the rapid clearance of

pDNA from the bloodstream in rats. The observed kinetics were found not to be

dependent upon plasmid sequence.













CHAPTER 4
DOSE DEPENDENCY OF PLASMID DNA PHARMACOKINETICS

Introduction

The studies presented in Chapters 2 and 3 have shown that degradation in the

plasma alone was not sufficient to describe the pharmacokinetics of pDNA. After IV

bolus administration SC pDNA was undetectable as early as 30 sec. This was in contrast

to isolated plasma when SC pDNA was detectable 3 minutes after the start of incubation

in isolated plasma. OC and L pDNA terminal half-life also decreased from 21 (1) to 5.3

(1.4) and 11 (2) to 1.9 (0.8) min, respectively.

Other investigators have suggested variable importance of plasma nucleases in the

degradation of genomic DNA after IV bolus administration. For example, Chused and

coworkers (Chused 1972), Whaley and Webb (Whaley 1972), and Tsumita and Iwanaga

(Tsumita 1963) all suggested a minimal role for plasma nucleases in the clearance of

DNA. This was based upon the observed fragmentation rate of genomic DNA in isolated

plasma versus the fragmentation rate after IV bolus administration, and the diffuse high

level of distribution of the DNA to tissues immediately after administration. This finding

was accompanied by the suggestion of extensive uptake of intact DNA molecules by the

reticuloendothelial system.

Alternatively, Gosse and coworkers (Gosse et al. 1965) found that "the plasma

DNases play a fundamental and probably exclusive role in the initial degradation of

DNA". This was based upon 3 observations. First was a rapid decrease in viscosity of








the blood within 3 minutes after administration. Second, this was based upon the

proportionality between the initial rate of degradation and the DNase activity level.

Third, this was also based upon the markedly slower disappearance of the DNA-methyl

green complex (a non-specific DNase inhibitor).

The disparity between the results presented here and the previous studies deserves

further investigation. Chused and coworkers, and Whaley and Webb, utilized smaller

doses of DNA in their experiments versus Gosse and coworkers (5 versus 200 jtg/

mouse). If this large dose had temporarily saturated an alternative clearance mechanism,

this would increase the observed importance of nucleases. Thus, nonlinear processes

may provide an explanation for the observed disparity.

Nonlinear clearance of pDNA has previously been suggested using

pharmacokinetic analysis of outflow patterns from rat perfused liver studies with

radiolabeled OC pDNA(Yoshida 1996). In this study, Vd increased and extraction ratio

decreased as perfusion dose was increased from 1.33 to 13.3 Pg/liver.

The purpose of this investigation was to model the pharmacokinetics of increasing

doses of naked pDNA in a topoform specific manner after IV bolus administration in the

rat. This information may provide an explanation for the disparity between the results

presented here and in previous studies. Furthermore, we sought to determine the

metabolite (OC and L) pharmacokinetics independently, by direct injection of each of the

metabolites. This information will provide a basis upon the percent conversion of the SC

to the OC form and the OC form to the L form of the plasmid.








Methods

Animals (male Sprague-Dawley rats 300-350 g) were purchased from Charles

River Laboratories (Wilmington, MA). Animals were housed in the University of Florida

Animal Resources Unit prior to all experiments and were given food and water ad

libitum. Animals were anesthetized by intraperitoneal injection with 0.5 ml of a cocktail

containing 13 mg/kg Xylazine, 2.15 mg/kg Acepromazine (The Butler Co., Columbus,

OH), and 66 mg/kg Ketamine (Fort Dodge Animal Health, Fort Dodge Iowa).

Phenol: chloroform: isoamyl alcohol (25: 24: 1, v/v/v), chloroform, Tris, boric

acid, EDTA, and agarose were purchased from Sigma Chemical Company (St. Louis,

MO). Ethidium bromide (electrophoresis grade) was purchased from Fisher Biotech

(Fair Lawn, NJ). Competent JM109 bacteria (Promega, Madison, WI) were transformed

according to the manufacturers directions with the pGL3 control plasmid (Promega,

Madison, WI) or pGeneMax plasmid (Gene Therapy Systems, San Francisco, CA).

Plasmid DNA was isolated from overnight cultures using alkaline lysis and

ultracentrifugation with a CsCl gradient (Katz 1977). Isolated pDNA was resuspended in

phosphate buffered saline. Plasmid was >90% SC by agarose gel analysis.

OC pDNA was produced by incubation of the SC pDNA, in phosphate buffered

saline, at 70'C for 16h. This procedure resulted in >90% OC plasmid (Figure 4-1). UV

absorbance at 260 nm and the A260/A280 ratio of the pDNA solution did not change

after this treatment (Figure 4-2).

L pDNA was produced by digestion with BamHI restriction enzyme (Promega,

Madison, WI) in separate reaction mixtures containing 173 utl of DI H20, 27 p.1 1 Ox

Buffer (Promega, Madison, WI) 56 p1 (100 ptg) pGL3, and 10 p.1 of BamHI (10 U/p.l).








The reaction mixture was incubated at 37C for 3 h. Plasmid was then isolated from the

reaction mixture by extraction with 1 volume of phenol: chloroform: isoamyl alcohol (25:

24: 1), followed by extraction with 1 volume of chloroform. Plasmid was then

concentrated by precipitation with 0.3 M Na Acetate, and 1 volume of isopropanol,

followed by centrifugation at 13K g for 30 min at 4C, and resuspended in 50 JAI of

phosphate buffered saline. This method routinely produced >90% L pDNA (Figure 4-3).

Concentration of pDNA was measured by monitoring UV absorbance at 260 nm, purity

was measured by A260/A280 ratio. A resulting purity of less than 1.7 was re-extracted

with 1 volume of chloroform until purity >1.7 was achieved.

For blood sampling, male Sprague-Dawley rats (300-350g) were anesthetized and

the jugular vein was exposed via an incision, isolated, ligated, and nicked with

ophthalmic scissors. A sterile silatstic (0.64 cm internal diameter by 0.12 cm outer

diameter, 10 cm in length) filled with sterile saline was threaded 30-40 mm into the

jugular vein and positioned just distal to the entrance to the right atrium and secured by

6.0 silk sutures. For injections, the femoral vein was isolated, and pDNA was injected

into the femoral vein using a 27-gauge needle. This method is graphically illustrated in

Figure 3-1 and 3-2. Isolated blood samples (approx. 300 tl) were drawn through the

jugular vein cannula and immediately placed in test tubes containing 0.57 ml of 0.34 M

EDTA (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) on ice at the times indicated.

This concentration of EDTA has previously been shown to inhibit the degradation of

pDNA in isolated rat plasma (Houk 1999).








2

















E-OC





: +SC











Figure 4-1 Agarose gel analysis of pDNA after conversion to the OC form of the plasmid.
Lane 1: Prior to treatment plasmid is predominately SC. Lane 2: After treatment plasmid
is completely converted to to OC form..





76
















3500


o 3000
0

z 2500
a.

2000


1500


1000
E
0

CL 500
0

C. 0
Supercoiled Open Circular

Figure 4-2. Absorbance of pDNA before and after conversion to the OC form. Data
represents averages of n=3 I standard deviation.








2 3 4















4-OC

-L






4-SC









Figure 4-3. Agarose gel analysis of pDNA before and after conversion to the L form of
the plasmid. Lane 1: Size standard, Lane 2: before treatment the plasmid is
predominately in the SC and OC form, Lane 3: Mixture of SC, OC, and L plasmid for
reference, Lane 4: after treatment the plasmid is completely converted to the L form.








To isolate pDNA from whole blood 250 il of blood was liquid/ liquid extracted

with 250 lal of phenol: chloroform: isoamyl alcohol (25: 24: 1, v/v/v), vortexed for 5 s at

low speed, and centrifuged at 20,800 g for 10 min at room temperature. The aqueous

phase was removed and stored at -20'C until analysis. Samples analyzed and quantitated

as described in Chapter 2.
Results

SC pDNA was detectable in the bloodstream only after a 2500 plg dose, no SC

pDNA was detectable in the bloodstream at lower doses as early as 30 sec after

administration. Because of this, the pharmacokinetic parameters reported for this form of

pDNA relied only on data acquired from this dose. SC pDNA remained detectable in the

plasma through 1 min after administration. Using the limited available data we

approximated that SC pDNA degraded with a half-life of 0.15 (0.01) min. The

degradation of SC pDNA was fit to a one-compartment body model with central

elimination and uptake (Figure 4-4). We extrapolated a least squares fit of the data to an

initial, t=0, concentration which was necessary as this area accounted for a major portion

of the AUC... Clearance of SC pDNA was calculated to be 390 (10) ml/min, and

volume of distribution was 148 (26) ml. (Table 4-1).

Concentrations of OC and L pDNA in the bloodstream after IV bolus

administration are displayed in Figure 4-5 and 4-6 respectively. Noncompartmental

analysis of all four doses is displayed in Table 4-2 for OC and Table 4-3 for L pDNA. A

decrease in terminal slope is observable with increasing dose for the OC form of the

plasmid (Figure 4-5). Clearance of the OC form of the plasmid decreased with increasing

dose (Table 4-2). Formation clearance values for the OC form of the plasmid after the








administration of SC pDNA ranged from 1.3 ( 0.2) to 8.3 ( 0.8) ml/min for the 2500,

and 250 jig doses respectively. Formation clearance of the L form of the pDNA

remained constant at an average of 6.7 ( 0.2) ml/min for all doses. The 250 jlg dose L

concentrations close to limits of quantitation, and thus required a large amount of

extrapolation for AUC calculation. For this reason, the 250 jig dose L analysis was

excluded from the noncompartmental analysis. Corresponding plots of OC pDNA

plasma concentrations, normalized for dose, were not superimposable (Figure 4-7)

(Gibaldi 1982).

To investigate the percent of SC plasmid that becomes OC as well as the percent

OC plasmid that becomes L, we compared the AUC obtained after IV bolus

administration of the OC and L forms of plasmid independently at 2500 and 250 lag

doses. Plasma concentrations of OC pDNA obtained after administration of 2500 and

250 lag doses are displayed in Figure 4-8 and 4-9 respectively. Noncompartmental

analysis of the OC form of the plasmid at each dose is displayed in Table 4-4. Clearance

again decreased between the 250 and 2500 jig doses 8.8 (2.4) to 1.3 (0.2) ml/min.

Clearance also remained consistent with that observed after administration of SC pDNA

at each dose, 8.8 ( 2.4) versus 8.3 (0.8) ml/ min at the 250 lag dose, and 1.3 ( 0.2)

versus 1.3 ( 0.2) at the 2500 jig dose. Volume of distribution of the OC form was 43

(15) ml.

Concentrations of L pDNA after administration of 2500 and 250 gig doses of L

pDNA are presented in Figure 4-10 and 4-11 respectively. Noncompartmental analysis is


















12


10-


8-

CD
-S.- 6T

z
CL4-


2

0T

0.0 0.5 1.0 1.5 2.0 2.

time (min)
Figure 4-4. Concentrations of SC pDNA in the bloodstream after 2500 lag dose. SC
pDNA remained detectable through 1 minute after administration. Data points represent
averages of n=3 I standard deviation. Lines represent a least squares fit of the data
using the model described in the Methods section.




















Table 4-1. Pharmacokinetic parameters estimated for supercoiled pDNA
fit t=0 concentration of SC pDNA


based upon the


Parameter Value

AUC (ng/ l*min) 6.4 (0.2)

MRT(min) 0.21 (0.02)

Cl (ml/min) 390 (10)

Vdss (ml) 148 (26)

Half-life (min) 0.15 (0.02)


Parameters represent averages of n=3 1 standard deviation.
















100

10


a
0.1
0 10 20 30 40 50 60
time (min)


Figure 4-5. Concentrations of OC pDNA after IV bolus administration of: U 2500 p.g, A
500 gg, @ 333 jtg, or 250 jtg of SC pDNA. Data represents mean of n=3.


















10




z
0
CL

0.1

0 10 20 30 40 50 60

time (min)
Figure 4-6. Concentrations of L pDNA after IV bolus administration of: U 2500 lg, A
500 lg, 0 333 gig, or 250 jig of SC pDNA. Data represents mean of n=3.



















Table 4-2. Noncompartmental analysis of OC pDNA after IV bolus administration of SC
pDNA.

Parameter 2500 tg 500 ptg 333 tg 250 Lg

Dose Dose Dose Dose

AUC 1200 120 (50) 59 (3) 18 (2)

(ng/jtl*min) (200)

AUC % extrapolated 1 (0.4) 9 (4) 10 (6) 24 (2)

AUMC 20000 1900 400 (20) 130 (20)

(ng/i1*min2) (6000) (1200)

MRT (min) 16 (3) 14 (3) 6.8 (0.4) 7.2 (0.3)

Cl/f (ml/min) 2.1 (0.4) 4.8 (2.0) 5.7 (0.3) 14 (1)

C1 (ml/min) 1.3 (0.2) 3.0 (1.2) 3.5 (0.2) 8.3 (0.8)

C. (ng/ tl) 49 (4) 13 (4) 6.5 (0.3) 2.2 (0.2)

tmax (min) 1 1 0.7 ( 0.3) 0.8 ( 0.3)


Parameters represent averages of n=3 ( 1 standard deviation).



















Table 4-3. Noncompartmental analysis of L pDNA after IV bolus administration of SC
pDNA.

Parameter 2500 [tg 500 pg 333 pg

Dose Dose Dose

AUC 240 (40) 52 (25) 32 (5)

(ng/jtl*min)

AUC % extrapolated 12 (7) 15 (5) 13 (7)

AUMC 7500 570 300 (20)

(ng/pl*min2) (2700) (370)

MRT (min) 31 (6) 10 (2) 9.6 (1.7)

Cl/f (ml/min) 10.6 (2.0) 11 (5) 11 (1)

Cl (ml/min) 6.5 (1.2) 6.9 (2.8) 6.6 (0.9)

Cmax (ng/tl) 5.4 ( 0.6) 3.2 (1.0) 2.4 (0.5)

t,,x (min) 22 ( 3) 5.3 ( 4.0) 6.0 ( 3.6)


Parameters represent averages of n=3 ( 1 standard deviation).




Full Text
PHARMACOKINETICS OF PLASMID DNA
By
BRETT EDWARD HOUK
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2000

Copyright 2000
by
Brett Edward Houk

This work is dedicated to my parents Nancy and Ronald Houk for all of their guidance
throughout my life.

ACKNOWLEDGMENTS
I would like to acknowledge Dr. Jeffrey A. Hughes who, aside from my parents,
has been the biggest influence in my life thus far. I would also like to acknowledge Dr.
Guenther Hochhaus for his invaluable insight and guidance in this work.
IV

TABLE OF CONTENTS
page
ACKNOWLEDGMENTS iv
LIST OF TABLES vii
LIST OF FIGURES ix
ABSTRACT xiv
INTRODUCTION 1
The Use of Naked pDNA as a Therapeutic Agent 2
Effectiveness of Naked Plasmid DNA after Local Administration 3
Effectiveness of Naked Plasmid DNA after IV Administration 6
Anatomical Factors Potentially Involved in the Pharmacokinetics of Plasmid DNA.. 9
Degradation of pDNA in the Bloodstream 16
Pharmacokinetics of Liposomal Delivery Vehicles 17
Pharmacokinetics of Naked Plasmid DNA and Liposome: Plasmid DNA Complexes
17
Pharmacokinetics of Naked Plasmid DNA in the Blood after IV Injection 17
Distribution of Plasmid DNA in Tissues after IV Injection 20
Conclusions 21
PHARMACOKINETICS OF PLASMID DNA IN ISOLATED RAT PLASMA 23
Introduction 23
Methods 25
Theoretical 35
Results 36
Conclusions 46
PHARMACOKINETICS OF PLASMID DNA AFTER IV BOLUS ADMINISTRATION
IN THE RAT 52
Introduction 52
Methods 53
Theoretical 57
Results 60
Conclusions 61
v

DOSE DEPENDENCY OF PLASMID DNA PHARMACOKINETICS
71
Introduction 71
Methods 73
Results 78
Conclusions 90
PHARMACOKINETIC MODELING OF PLASMID DNA AFTER IV BOLUS
ADMINISTRATION IN THE RAT 102
Introduction 102
Theoretical 104
Results 106
Conclusions 114
PHARMACOKINETICS OF LIPOSOME: PLASMID DNA COMPLEXES 122
Introduction 122
Methods 125
Results 127
Pharmacokinetics of Liposome:pDNA Complex Degradation in Rat Plasma... 127
Pharmacokinetics of Liposome:pDNA Complex Degradation in Rat Whole Blood
128
Pharmacokinetics of Liposome:pDNA Complexes after IV Bolus Administration
in the Rat 129
Conclusions 130
CONCLUSIONS AND IMPLICATIONS 145
Summary of Results 145
Implications of Plasmid DNA Degradation in Isolated Plasma 145
Comparison of In Vitro and In Vivo Pharmacokinetics 146
Effects of Increasing Dose of Plasmid DNA 148
Results of the Curve Fitting Experiments 151
Liposome: pDNA Complex Conclusions 152
Future Directions 155
Concluding Remarks 157
LIST OF REFERENCES 159
BIOGRAPHICAL SKETCH 166
vi

LIST OF TABLES
Table page
2-1. Method parameters for pDNA analysis 34
2-2. Pharmacokinetic parameters for pDNA in isolated rat plasma 41
2-3. Noncompartmental parameters for pDNA after incubation in isolated plasma 42
2-4. Pharmacokinetic parameters for pDNA after incubating the pGE150 plasmid in
isolated rat plasma 49
3-1. Pharmacokinetic parameters calculated after 500 pg dose of SC pDNA 65
3-2. Comparison of in vivo and in vitro pharmacokinetic parameters for pDNA 66
3-3. Comparison of OC and L pDNA parameters after administration of SC pGL3,
pGE150, and pGeneMax 69
4-1. Pharmacokinetic parameters estimated for supercoiled pDNA based upon the fit t=0
concentration of SC pDNA 81
4-2. Noncompartmental analysis of OC pDNA after IV bolus administration of SC
pDNA 84
4-3. Noncompartmental analysis of L pDNA after IV bolus administration of SC pDNA. .85
4-4. Noncompartmental analysis of OC pDNA after IV bolus administration of OC
pDNA at 2500 and 250 pg doses 89
4-5. Noncompartmental analysis of L pDNA after IV bolus administration of L pDNA at
2500 and 250 pg doses 93
5-1. Pharmacokinetic parameters for pDNA based upon the model presented in the text. ..112
5-2. Overall pharmacokinetic parameters for pDNA when all doses are fit
simultaneously 113
vii

5-3. Pharmacokinetic parameters calculated after administration of OC pDNA at 2500
and 250 pg doses 118
5-4. Pharmacokinetic parameters calculated after administration of L pDNA at 2500 and
250 pg doses 119
6-1. Noncompartmental analysis of pDNA after administration of liposome: SC pDNA
complexes 137
6-2. Comparison of SC pDNA pharmacokinetic parameters after administration of SC
pDNA either in free form (naked) at 2500 pg dose or after administration as
liposome: pDNA complexes at 500 pg dose 138
6-3. Comparison of OC pDNA pharmacokinetic parameters after administration of SC
pDNA either in free form (naked) or after administration as liposome: pDNA
complexes at 500 pg pDNA dose 139
6-4. Comparison of L pDNA pharmacokinetic parameters after administration of SC
pDNA either in free form (naked) or after administration as liposome: pDNA
complexes at 500 pg pDNA dose 140
viii

LIST OF FIGURES
Figure page
1 -1. Potential sights for nicking of the phosphodiester backbone of DNA 10
1-2. Model of plasmid DNA degradation in the bloodstream 11
1-3. Schematic representation of pDNA (•) passing through a continuous capillary: (1)
pinocytosis, (2) through intercellular junctions, and (3) passing through
endothelial channels 12
1-4. Schematic representation of pDNA (•) passing through a fenestrated capillary: (1)
pinocytosis, (2) passing through a diaphragm fenestrae, and (3) passing through
and open fenestrae 13
1-5. Schematic representation of pDNA (•) passing through a discontinuous capillary.
(1) pinocytosis and (2) passing through large pores in the endothelium 14
2-1. Plasmid map of pGL3 Control 26
2-2. Plasmid map of the pGeneMax-Luciferase 27
2-3. Plasmid map of pGE 150 28
2-4. Standard curve for supercoiled pDNA. Abcissa represents total ng estimated by UV
absorbance at 260 nm. Ordinate represents ng calculated by the KDS ID
software. Error bars represent ±1 standard deviation 31
2-5. Standard curve for OC pDNA. Abcissa represents total ng estimated by UV
absorbance at 260 nm. Ordinate represents ng calculated by the KDS ID
software. Error bars represent ±1 standard deviation 32
2-6. Standard curve for Linear pDNA. Abcissa represents total ng estimated by UV
absorbance at 260 nm. Ordinate represents ng calculated by the KDS ID
software. Error bars represent ±1 standard deviation 33
2-7. Pharmacokinetic model of plasmid DNA degradation in rat plasma. The model is
considered to be a unidirectional process. SC, OC, and L represent the amounts
of supercoiled, open circular, and linear plasmid, respectively, in each
IX

compartment. The rate constants ks, k0, and ki represent the degradation constants
for supercoiled, open circular, and linear plasmid, respectively 37
2-8. Agarose gel analysis of pDNA degradation in isolated rat plasma. Lane 1; size
standard, lane 2; 30 sec, lane 3; 1 min, lane 4; 2 min, lane 5; 3 min, lane 6; 5 min,
lane 7; 10 min, lane 8; 20 min, lane 9; 30 min, lane 10; 45 min, lane 11; 60 min,
lane 12; 80 min 39
2-9. Experimental and fitted data based on the pharmacokinetic model described in the
text. Data points represent actual experimental data. Lines represent values
predicted by the model. Data represents mean of n=3 ± 1 standard deviation.
Key: ® supercoiled, • open circular, ▲ linear 40
2-10. Analysis of rate constants in dilute plasma. Degradation rate constants were
modeled in PBS diluted rat plasma. Rate constants represent the fitted values of
n=6 rats/time point. Key: ♦ks in dilute plasma, Bko in dilute plasma. The value
of ki is not reported due to the prolonged stability of linear plasmid in dilute
plasma 44
2-11. Degradation of supercoiled pDNA in 25% rat plasma after (A) incubating the
plasma at 90°C for 10 min (B) the addition of 0.1 mM EDTA 45
2-12. Comparison of concentrations of OC pDNA using (♦) pGE150 concentrations of
OC pDNA using (â– ) pGL3 in isolated rat plasma. Data represents mean of n=3
±1 standard deviation 47
2-13. Comparison of concentrations of L pDNA using (♦) pGE150 versus
concentrations of L pDNA using (â– ) pGL3 in isolated rat plasma. Data
represents mean of n=3 ±1 standard deviation 48
3-1. Photograph of the jugular cannula placement used for blood sampling 55
3-2. Photograph of the femoral vein isolation and injection procedure used for IV bolus
administration 56
3-3. A representative gel from which plasmid amounts were quantified as described in
the methods section. Lane 1: size standard, lane 2: 1 min, lane 3: 2 min, lane 4:
3.5 min, lane 5: 5 min, lane 6: 10 min, lane 7: 20 min, lane 8: 30 min, lane 9: 45
min, lane 10: 60 min 59
3-4. Experimental and fitted data based on the pharmacokinetic model described in the
text. Data points represent actual experimental data. Lines represent values
predicted by the model. Data represents mean of n=6 + 1 standard devaition.
Key: • open circular, A linear 62
3-5. Concentrations of OC pGL3 after â– : IV bolus administration of a 500 pg dose of
SC pGL3, and ♦: Incubation of SC pGL3 in isolated plasma at 37°C 63
x

3-6. Concentrations of L pGL3 after â– : IV bolus administration of a 500 pg dose of SC
pGL3, and ♦: Incubation of SC pGL3 in isolated plasma at 37°C 64
3-7. Concentrations of OC pDNA in the bloodstream after IV bolus administration of
500 pg of pCMV-Luc, pGE150, or pGL3. Data represents mean of n=3 ± 1
standard deviation 67
3-8. Concentrations of L pDNA in the bloodstream after IV bolus administration of 500
pg of pCMV-Luc, pGE150, or pGL3. Data represents mean of n=3 ± 1 standard
deviation 68
4-1 Agarose gel analysis of pDNA after conversion to the OC form of the plasmid. Lane
1: Prior to treatment plasmid is predominately SC. Lane 2: After treatment
plasmid is completely converted to to OC form 75
4-2. Absorbance of pDNA before and after conversion to the OC form. Data represents
averages of n=3 ± 1 standard deviation 76
4-3. Agarose gel analysis of pDNA before and after conversion to the L form of the
plasmid. Lane 1: Size standard, Lane 2: before treatment the plasmid is
predominately in the SC and OC form, Lane 3: Mixture of SC, OC, and L
plasmid for reference, Lane 4: after treatment the plasmid is completely converted
to the L form 77
4-4. Concentrations of SC pDNA in the bloodstream after 2500 pg dose. SC pDNA
remained detectable through 1 minute after administration. Data points represent
averages of n=3 ± 1 standard deviation. Lines represent a least squares fit of the
data using the model described in the Methods section 80
4-5. Concentrations of OC pDNA after IV bolus administration of: â–  2500 pg, â–² 500
pg, • 333 pg, or ♦ 250 pg of SC pDNA. Data represents mean of n=3 82
4-6. Concentrations of L pDNA after IV bolus administration of: â–  2500 pg, â–² 500 pg,
• 333 pg, or ♦ 250 pg of SC pDNA. Data represents mean of n=3 83
4-7. Superposition of OC pDNA concentrations normalized for dose after administration
of •: 2500 pg, ▲: 500 pg, ♦: 333 pg, or ^250 pg dose. Data represents mean
ofn=3 ± 1 standard deviation 86
4-8. Concentrations of OC pDNA in the bloodstream after administration of OC pDNA
at a 2500 pg dose. Data represents mean of n=3 ± 1 standard deviation 87
4-9. Concentrations of OC pDNA in the bloodstream after administration of OC pDNA
at a 250 pg dose. Data represents mean of n=3 ± 1 standard deviation 88
4-10. Concentrations of L pDNA in the bloodstream after administration of L pDNA at a
2500 pg dose. Data represents averages of n=3 ± 1 standard deviation 91
xi

4-11. Concentrations of L pDNA in the bloodstream after administration of L pDNA at a
250 pg dose. Data represents averages of n=3 ± 1 standard deviation 92
4-12. Area under the curve of OC pDNA after administration of a 2500 pg dose of SC or
OC pDNA. Data represents mean of n=3 ± 1 standard deviation. * Indicates
statistical significance by one way ANOVA (p<0.05) 94
4-13. Area under the curve of OC pDNA after administration of a 250 pg dose of SC or
OC pDNA. Data represents mean of n=3 ± 1 standard deviation. * Indicates
statistical significance by one way ANOVA (p<0.05) 95
4-14. Area under the curve of L pDNA after administration of a 2500 pg dose of SC or
OC pDNA. Data represents mean of n=3 ± 1 standard deviation. * Indicates
statistical significance by one way ANOVA (p<0.05) 96
4-15. Area under the curve of L pDNA after administration of a 250 pg dose of SC or
OC pDNA. Data represents mean of n=3 ± 1 standard deviation. AUC
differences were not statistically significant by one way ANOVA 97
5-1. Model for pDNA clearance from the bloodstream 105
5-2. Concentrations of (A) OC and (B) L pDNA in the bloodstream after 2500 pg dose
of SC pDNA. Data points represent the averages of n=3 ±1 standard deviation.
Lines represent concentrations predicted by the model 108
5-3. Concentrations of (A) OC and (B) L pDNA in the bloodstream after 500 pg dose of
SC pDNA. Data points represent the averages of n=3 ±1 standard deviation.
Lines represent concentrations predicted by the model 109
5-4. Concentrations of (A) OC and (B) L pDNA in the bloodstream after 333 pg dose of
SC pDNA. Data points represent the averages of n=3 ±1 standard deviation.
Lines represent concentrations predicted by the model 110
5-5. Concentrations of (A) OC and (B) L pDNA in the bloodstream after 250 pg dose of
SC pDNA. Data points represent the averages of n=3 ±1 standard deviation.
Lines represent concentrations predicted by the model Ill
5-6. Concentrations of OC pDNA in the bloodstream after (A) 2500 pg and (B) 250 pg
dose of OC pDNA. Data points represent the averages of n=3 ±1 standard
deviation. Lines represent concentrations predicted by the model 115
5-7. Concentrations of L pDNA in the bloodstream after (A) 2500 pg and (B) 250 pg
dose of OC pDNA. Data points represent the averages of n=3 ±1 standard
deviation. Lines represent concentrations predicted by the model 116
xii

5-8. Concentrations of L pDNA in the bloodstream after (A) 2500 pg and (B) 250 pg
dose of L pDNA. Data points represent the averages of n=3 ± 1 standard
deviation. Lines represent concentrations predicted by the model 117
6-1. Liposome-pDNA complexes were incubated in rat plasma for various time points.
10 pi of sample was loaded in each lane as described in the methods section.
Lane 1; size standard, lane 2; 1 min, lane 3; 2 min, lane 4; 5 min, lane 5; 10 min,
lane 6; 20 min, lane7; 30 min, lane 8; 60 min, lane9; 2 h, lane 10; 3 h, lane 11; 5.5
h 131
6-2. Agarose gel analysis of liposome/pDNA complexes. (A) 1:1 lipid:pDNA ratio,
through 4 hours. (B) 3:1 lipid:pDNA ratio, through 6 hours. (C) 6:1 lipid:pDNA
ratio, through 6 hours, indicates the 3 hour time point 132
6-3. Lane 1: high molecular weight size standard, lane 2: 1:1 lipid:pDNA complexes,
lane 3: 3:1 lipid:pDNA ratio (w/w), lane 4: 6:1 lipid:pDNA ratio 133
6-4. (A)Degradation of SC pDNA in rat blood versus plasma. (B)Degradation of
supercoiled pDNA in 3:1 and 6:1 (w/w) liposome/pDNA complexes incubated in
heparinized rat whole blood. Error bars indicate standard deviation of n=3 rats 134
6-5. Agarose gel analysis of pDNA after administration of liposome: pDNA complexes.
Lane 1: 15 sec, lane 2: 30 sec, lane 3: 45 sec, lane 4: 1 min, lane 5: 1.5 min, lane
6: 2 min, lane 7: 2.5 min, lane 8: 3 min, lane 9: 4 min, lane 10: 5 min 135
6-6. Plasma concentrations of SC, OC, and L pDNA after 500 pg IV bolus
administration of SC pDNA: liposome complexes. Key: ♦: SC, ■: OC, ▲: L 136
7-1. Schematic representation of pDNA degradation in isolated plasma 147
7-2. Schematic representation of pDNA pharmacokinetic parameters after IV bolus
administration of SC pDNA in the rat 154
7-3. Schematic representation of liposome pDNA clearance from the bloodstream. In
this model, removal from the bloodstream of the lipid: pDNA complexes is
assumed to be larger than the degradation of the complex 156
xiii

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
PHARMACOKINETICS OF PLASMID DNA
By
Brett E. Houk
May 2000
Chairman: Dr. Jeffrey A. Hughes
Cochairman: Dr. Guenther Hochhaus
Major Department: Pharmaceutics
We sought to construct a complete pharmacokinetic model to describe the
degradation of all three topoforms, supercoiled (SC), open circular (OC), and linear (L),
of pDNA in vivo and in vitro. SC pDNA was incubated in isolated rat plasma at 37°C in
vitro. At various time points, the plasma was assayed by electrophoresis for the amounts
of SC, OC, and L pDNA remaining. The calculated amounts remaining were fit to linear
differential equations describing this process. The calculated pharmacokinetic
parameters suggested that SC pDNA degrades in isolated rat plasma with a half-life of
1.2 min, OC pDNA degrades with a half-life of 21 min, and L pDNA degrades with a
half-life of 11 min. Complexation of pDNA with cationic liposomes resulted in a portion
of the supercoiled plasmid remaining detectable through 5.5 h in vitro. We next
investigated the pharmacokinetics of SC plasmid DNA after IV bolus administration in
the rat by following SC, OC, and L pDNA. SC pDNA was detectable in the bloodstream
only after the highest, 2500 pg, dose and had a clearance of 390(±10) ml/min and volume
xiv

of distribution of 148(±26) ml. The pharmacokinetics of OC pDNA exhibited non-linear
characteristics with clearance ranging from 8.3(±0.8) to 1.3(±0.2) ml/min and a volume
of distribution of 39(±19) ml. L pDNA exhibited linear kinetics and was cleared at
7.6(±2.3) ml/min with a volume of distribution of 37(±17) ml. AUC analysis revealed
60(±10) % of the SC was converted to the OC form, and nearly complete conversion of
the OC pDNA to L pDNA. Clearance of SC pDNA was decreased after liposome
complexation to 87(±30) ml/min. However, the clearance of OC and L pDNA was
increased relative to naked pDNA at an equivalent dose to 37(±9) ml/min and 95(±37)
ml/min, respectively. We conclude that SC pDNA is rapidly cleared from the circulation
OC pDNA displays non-linear pharmacokinetics. L pDNA exhibits first order kinetics.
Liposome complexation protects the SC topoform, but the complexes are more rapidly
cleared than the naked pDNA.
xv

CHAPTER 1
INTRODUCTION
Biotechnology is one of the most rapidly growing areas in the pharmaceutical
sciences today. However, biotechnology products (e.g. proteins and peptides) suffer
from poor stability, low absorption, and difficulties in delivery. It would therefore be
ideal if the protein could be made in vivo, utilizing the body’s own mechanisms to
produce the competent protein. Gene therapy is one potential route by which to
accomplish this goal. Gene therapy also offers the potential treatment of genetic
diseases. The replacement of mutated, missing, or deleted DNA via gene therapy can
result in the production of a competent protein. These potentials make gene therapy one
of the most exciting and rapidly advancing areas of biotechnology.
Early studies have revealed that systemically administered plasmid DNA (pDNA)
can be expressed in animals (Kawabata et al. 1995; Mahato et al. 1995; Osaka et al. 1996;
Song et al. 1997; Thierry et al. 1997) and humans (Valere 1999). Intravenous (IV)
administration of DNA offers the potential advantage of allowing a wide distribution of
activity in the body (Lew et al. 1995; Thierry 1995; Osaka et al. 1996; Thierry et al.
1997). This route of administration allows the treatment of non-localized and systemic
diseases. Previous research on the pharmacokinetics of non-viral gene therapies have
only been observational citing that plasmid DNA degrades within 5 minutes after
incubation in whole blood in vitro or after IV injection (Kawabata et al. 1995; Thierry et
al. 1997).
1

2
Plasmid DNA exists as three major topoforms. The native structure of non-
damaged pDNA is supercoiled (SC). Single strand nicks to the phosphodiester backbone
of pDNA yield an open circular (OC) form (Figure 1-1). This metabolite of SC pDNA is
associated with significant transcriptional activity (-90-100%) (Adami et al. 1998; Niven
et al. 1998). Further single strand nicks to the OC pDNA yield linear (L) pDNA,
associated with a significant loss of activity (-90%). This process is schematically
illustrated in Figure 1-2.
In order to properly dose and achieve the desired levels of gene expression it will
be necessary to understand the pharmacokinetics of pDNA. In initial human clinical
trials with viral gene therapy, at least one study was terminated due to a patient death
(Press 1999). This death was later attributed to the high doses utilized in the trails. Thus,
the pharmacokinetics of pDNA is an essential area to be considered as gene therapy
approaches clinical use.
The Use of Naked pDNA as a Therapeutic Agent
The use of naked pDNA as a drug after intravenous (IV) administration has been
intensely investigated (Wang et al. 1995; Takeshita et al. 1996; Zhang et al. 1997; Budker
et al. 1998; Song et al. 1998; Wang et al. 1998; Witzenbichler et al. 1998; Liu 1999;
Zhang et al. 1999). The use of naked pDNA in vivo was initially reported after
intramuscular (IM) or intradermal (SQ) administration in mammals (Wolff et al. 1991;
Fazio et al. 1994; Katsumi et al. 1994; Bright et al. 1995; Donnelly et al. 1995; Lopez-
Macias et al. 1995; Ulmer et al. 1995; Bright et al. 1996; Corr et al. 1996; Casares et al.
1997; Danko et al. 1997; Lawson et al. 1997; Ragno et al. 1997; Haensler et al. 1999;
Noll et al. 1999; Osorio et al. 1999). These studies have definitively shown efficient
expression of a transgene can be achieved after administration of naked pDNA. The

3
successes in these studies suggest that pharmacokinetic modeling of pDNA in the
bloodstream after IV administration, or pDNA appearing in the bloodstream after local
administration, is an area that must be more clearly defined in order to optimize gene
therapy for clinical use.
Effectiveness of Naked Plasmid DNA after Local Administration
Fazio and coworkers (Fazio et al. 1994) demonstrated that a transgene could be
efficiently secreted into the circulation after IM administration. Plasma accumulation of
human Apo-E2 was demonstrated for at least 45 days after injection. After
administration of pDNA encoding for an interferon transgene, interferons were detected
from days 7 to 28 post-DNA innoculation (Lawson et al. 1997). Administration of
plasmid DNA encoding the chloramphenicol acetyltransferase gene (CAT) in sterile
water lead to CAT transgene expression that peaked between 1 and 3 days and was
detected up to 28 days after DNA administration. Together these results indicate that
sustained expression can be obtained.
Efficient immunization of monkeys, mice, dogs, and cats has been demonstrated
using naked pDNA (Katsumi et al. 1994; Lopez-Macias et al. 1995; Ulmer et al. 1995;
Bright et al. 1996; Ragno et al. 1997; Haensler et al. 1999; Noll et al. 1999; Osorio et al.
1999). After injection of naked pDNA encoding for influenza hemagglutinin into the
skin of mice and monkeys, induction of significant ELISA antibody titers and
hemagglutination (HA) inhibition titers that were above the usual threshold values
predictive of protection against influenza were demonstrated (Haensler et al. 1999). Mice
immunized by various mucosal routes with a pDNA carrying the HA gene (pVlj- HA)
induced a HA-specific cytotoxic T lymphocyte (CTL) response. Similarly, nasal

4
immunization with the DNA vaccine induced primary CTLs against measles virus HA
(Etchart et al. 1997).
Plasmid DNA may also serve as an attractive means by which immunization to
parasitic infection may be achieved. After injection of pDNA encoding for heat shock
protein 65, T cell proliferation and antibodies to this protein were found to be elevated in
rats when compared with both an arthritic control and naive animals (Ragno et al. 1997).
A single immunization with pDNA encoding for Yersinia enterocolitica 60-kDa heat
shock protein (Y- HSP60) was used for vaccination and induced significant Y-HSP60-
specific T cell responses after 1 week (Noll et al. 1999). Induction of antibodies against
Salmonella typhi OmpC porin by naked DNA immunization has also been demonstrated
(Lopez-Macias et al. 1995).
A pDNA expression vector encoding human factor IX as an example of
immunogen was injected into mice three times at 10-day intervals (Katsumi et al. 1994).
This resulted in production of antibodies to human factor IX. Spleen cells from
inoculated mice also showed significant cytotoxic T lymphocyte response to target cells
expressing human factor IX. Thus, IM and SQ injection of pDNA can induce immune
responses against the encoded protein without an exposure to virus particles, and this
approach may serve as the basis for immunotherapy in the treatment of cancer and
infectious diseases in humans.
Plasmid DNA encoding for viral proteins is also an attractive means by which
immunization to viral infection may be achieved. The applicability of pDNA
immunization technology for vaccine development was also investigated by immunizing
dogs and cats by the IM and SQ routes with a pDNA vector encoding the rabies virus

5
glycoprotein G (Osorio et al. 1999). The results demonstrated that non-facilitated, naked
pDNA vaccines can elicit strong, antigen-specific immune responses in dogs and cats,
and DNA immunization may be a useful tool for future development of novel vaccines
for these species. Plasmid DNA encoding for the large tumor antigen (T- Ag) of SV40
was used to actively immunize mice to assess the induction of SV40 T-Ag-specific
immunity (Bright et al. 1996). Direct injection of the recombinant SV40 T-Ag protein
alone failed to induce SV40 T-Ag-specific CTL responses, whereas the pDNA encoding
SV40 T-Ag elicited CTL activity specific for SV40 T-Ag. Naked pDNA induced
immune responses that were protective against a lethal challenge with SV40-transformed
cells.
Naked pDNA has also been successful in the treatment of cancer by local
administration. Direct intratumoral injection of free pDNA into mouse melanoma BL6
solid tumor can also result in a high level of transfection. The average amount of
chloramphenicol acetyltransferase (CAT) expressed by injecting 30 pg pDNA containing
a CAT gene into a single BL6 tumor was 1.9 +/- 1.0 ng, which is comparable to that
reported in the skeletal muscle (Yang and Huang 1996). An intratumoral injection of
naked pDNA containing the HSV-TK gene (pAGO) resulted in tumor weight reduction
(40-50%) in treated animals versus control groups. Moreover, histopathological analysis
on tumors showed large areas of cavitary necrosis (85%) in treated groups compared to
controls (10%) (Soubrane et al. 1996). Thus direct injection of free pDNA may offer a
simple and effective approach and might be a potential method for cancer gene therapy.

6
Effectiveness of Naked Plasmid DNA after IV Administration
Naked pDNA administration by IV injection has also been shown to be an
effective means by which high levels of gene expression can be obtained (Wang et al.
1995; Takeshita et al. 1996; Zhang et al. 1997; Budker et al. 1998; Song et al. 1998;
Wang et al. 1998; Witzenbichler et al. 1998; Liu 1999; Zhang et al. 1999). Budker and
coworkers demonstrated that pDNA can be delivered to and expressed within skeletal
muscle of rats when injected rapidly, in a large volume (2 to 3 ml) (Budker et al. 1998).
Liu and coworkers also showed naked pDNA can be efficiently expressed in mice
(Liu 1999). As high as 45 pg of luciferase protein per gram of liver could be recovered
by a single tail vein injection of 5 pg of naked pDNA. Approximately 45% of
hepatocytes expressed the transgene. Peak expression was obtained at 8 hours after
administration and could be retained with repeated injections.
Efficient naked pDNA expression has been obtained following delivery via the
portal vein, hepatic vein, bile duct or direct IV administration via the tail vein in mice,
rats, and dogs (Zhang et al. 1997; Zhang et al. 1999). The highest levels of expression
were achieved after IV administration by rapidly injecting the pDNA in large volumes,
approximately 2.5 ml. Over 15 pg of luciferase protein/liver was produced in mice and
over 50 pg in rats. Equally high levels of beta-galactosidase (beta-Gal) expression were
obtained, in over 5% of the hepatocytes that had intense blue staining. Expression of
luciferase or beta-Gal was evenly distributed in hepatocytes throughout the entire liver
when either of the three routes were injected. Peri-acinar hepatocytes were preferentially
transfected when the portal vein was injected in rats. These levels of foreign gene
expression are among the highest levels obtained with nonviral vectors. Repetitive

7
pDNA administration through the bile duct led to sustianed foreign gene expression.
This study demonstrates that high levels of pDNA expression in hepatocytes can be
easily obtained by IV injection.
Takeshita and coworkers (Takeshita et al. 1996) investigated the hypothesis that
naked pDNA encoding for vascular endothelial growth factor (VEGF) could be used in a
strategy of arterial gene therapy to stimulate collateral artery development. Plasmid
DNA encoding each of the three principle human VEGF isoforms (phVEGF121,
phVEGF165, or phVEGF189) was applied to the hydrogel polymer coating of an
angioplasty balloon and delivered percutaneously to one iliac artery of rabbits with
operatively induced hindlimb ischemia. Compared with control animals transfected with
LacZ, site-specific transfection of phVEGF resulted in augmented collateral vessel
development documented by serial angiography, improvement in calf blood pressure
ratio (ischemic to normal limb), resting and maximum blood flow, and capillary to
myocyte ratio (suggesting increased vascularization). Similar results were obtained with
phVEGF121, phVEGF165, and phVEGF189. This suggests that these isoforms are
biologically equivalent with respect to in vivo angiogenesis. The potential for VEGF-C
to promote angiogenesis in vivo was then tested in a rabbit ischemic hindlimb model
(Witzenbichler et al. 1998). Ten days after ligation of the external iliac artery, VEGF-C
was administered as naked pDNA (pcVEGF-C; 500 pg) from the polymer coating of an
angioplasty balloon or as recombinant human protein (rhVEGF-C; 500 pg) by direct
intra-arterial infusion. Physiological and anatomical assessments of angiogenesis 30 days
later showed evidence of therapeutic angiogenesis for both pcVEGF-C and rhVEGF-C.
Hindlimb blood pressure ratio (ischemic/normal) after pcVEGF-C increased after

8
pcVEGF-C versus controls and after rhVEGF-C versus control rabbits receiving rabbit
serum albumin. Doppler- derived iliac flow reserve was increased for pcVEGF-C versus
controls and increased for rhVEGF-C versus albumin controls. Neovascularity was
documented by angiography in vivo after administration of pcVEGF-C and capillary
density was measured at necropsy increased. Arterial gene transfer of naked pDNA
encoding for a secreted angiogenic cytokine, thus, represents a potential alternative to
recombinant protein administration for stimulating collateral vessel development.
Naked pDNA constructs encoding for the human kallikrein protein delivered to
spontaneously hypertensive rats via IV injection have been shown to be efficient at
controlling hypertension (Wang et al. 1995). The expression of human tissue kallikrein in
rats was identified in the heart, lung, and kidney by reverse transcription polymerase
chain reaction followed by Southern blot analysis and an ELISA specific for human
tissue kallikrein. A single injection of both human kallikrein pDNA constructs caused a
sustained reduction of blood pressure, which began 1 week after injection and continued
for 6 weeks. A maximal effect of blood pressure reduction of 46 mm Hg in rats was
observed 2-3 weeks after injection with kallikrein pDNA as compared to rats with vector
pDNA. These results show that direct gene delivery of human tissue kallikrein causes a
sustained reduction in systolic blood pressure in genetically hypertensive rats and
indicate that the feasibility of kallikrein gene therapy for treating human hypertension
should be studied.
Collectively, these results suggest that IV administration of naked pDNA is an
attractive means to treat a large range of diseases. However, complete pharmacodynamic
modeling of pDNA will has not been achieved. This will allow correlation of the

9
administered dose with the desired levels of gene expression at the site of activity.
Because of the plasmids high molecular weight, anatomical factors must be considered in
the movement of these molecules within the body.
Anatomical Factors Potentially Involved in the Pharmacokinetics of Plasmid DNA
Plasmid DNA is a macromolecule having a molecular weight of 3.5 million for a
typical plasmid of 5.5 kilobase pairs. This large molecular weight results in an increased
likelihood of clearance processes being a function of size and its resulting abitily to pass
through capillary endothelia. After IV administration, distribution of macromolecules is
limited by the structure of the vascular endothelium. The structure of capillaries is
diverse among organs. There are 3 main types of blood capillaries: continuous,
fenestrated, and discontinuous (Hwang et al. 1997; Takakura et al. 1996).
These 3 types of capillaries are represented in Figures 1-3, 1-4, and 1-5. The
diameter of the free plasmid varies from between 8 to 22 nm (Yarmola 1985). The
passage of pDNA through a continuous capillary would be limited to the 50 nm
pinocytotic vesicles, 2 to 6 nm intracellular junctions, and 50 nm transendothelial
channels (Figure 1-3) (Hwang et al. 1997). The basal lamina presents a barrier of
collagen, glycoproteins, and fibronectin, macromolecules greater than 11 nm can be
retained by the basal lamina. Thus, this may present a barrier for diffusion of the plasmid
(Hwang et al. 1997). Continuous capillaries are the most widely distributed in
mammalian tissue and are found in skeletal, cardiac, and smooth muscles, as well as lung,
skin, subcutaneous tissues, serous membranes, and mucus membranes (Takakura et al.
1996).

10
Figure 1-1. Potential sights for nicking of the phosphodiester backbone of DNA.

11
Endonuclease action: Endonuclease action: Endonuclease or
Single strand nick to Single strand nick exonuclease action
the plasmid adjacent to previous
Figure 1-2. Model of plasmid DNA degradation in the bloodstream.

12
1
2
3
50 nm
2-6 nm
50 nm
Figure 1-3. Schematic representation of pDNA (•) passing through a continuous
capillary: (1) pinocytosis, (2) through intercellular junctions, and (3) passing through
endothelial channels.

13
Figure 1-4. Schematic representation of pDNA (•) passing through a fenestrated
capillary: (1) pinocytosis, (2) passing through a diaphragm fenestrae, and (3) passing
through and open fenestrae.

14
1 2
• •
50 nm 102-103 nm
Figure 1-5. Schematic representation of pDNA (•) passing through a discontinuous
capillary. (1) pinocytosis and (2) passing through large pores in the endothelium.

15
Fenestrated capillaries (Figure 1-4) are more likely to allow passage of pDNA
into tissues. The pDNA may be transported through mechanisms similar to those
involved in the continuous capillary, in addition to transport through 20 to 60 nm
fenestrae (Hwang et al. 1997; Takakura et al. 1996). These fenestrae may or may not be
closed by a diaphragm. The diameter of the closed diaphragm has not been reported.
This type of capillary is generally found in the intestinal mucosa, endocrine glands,
exocrine glands, glomerulus, and peritubular capillaries (Takakura et al. 1996).
Discontinuous capillaries are characterized by endothelial gaps and large pores
with diameters ranging from 100 to 1000 nm (Figure 1-5) (Hwang et al. 1997; Takakura
et al. 1996). In these capillaries there is little restriction of diffusion of macromolecules.
Another characteristic of this type of capillary is the lack of a basal lamina (Hwang et al.
1997). The mucopolysaccharide rich interstitial Spaces of Disse have pore diameters
ranging from 36 to 50 nm and are unlikely to present a major barrier for the transport of
pDNA. The discontinuous capillary is more limited in its distribution than the other
types and is found only in the liver, spleen, and bone marrow (Takakura et al. 1996).
These anatomical features can play an important role in the distribution of IV
administered pDNA, and other macromolecules. In addition, capillary permeability can
be further enhanced in pathophysiological states such as cancer and inflammation
(Takakura et al. 1996). Thus the fate of IV administered pDNA is determined not only
by physio-chemical properties such as molecular weight, but also by anatomical features
of the capillary endothelium present in each tissue.

16
Degradation of pDNA in the Bloodstream
Early studies suggested that serum nucleases play a major role in the clearance of
DNA from the bloodstream of injected animals (Gosse et al. 1965). Investigations by
Chused and coworkers suggested that nucleases may not play a major role in the
degradation of tritiated KB cell genomic DNA when IV injected in mice (Chused 1972).
However, their assay was not able to identify the true activity of nucleases given that
their assay utilized genomic DNA. Single strand cuts to the isolated genomic DNA
would not yield small fragments and would be undetectable by their method. This would
yield an underestimation of true nuclease activity. In contrast, single strand cuts to
pDNA would lead to a degradation of the native SC structure to the OC form of the
plasmid and be detectable by agarose gel analysis.
Nucleases represent two subclasses of enzymes, endonucleases and exonucleases.
Endonucleases act on the phosophodiester backbone of DNA in a continuous chain
(Lodish 1995). Whereas, endonucleases act upon the free end (5’ or 3’) of the
phosphodiester backbone in a linear segment of DNA. Investigations by Thierry and
coworkers , utilizing agarose gel analysis, suggested that the main nuclease activity in the
bloodstream was endonucleolytic. This was based on the finding that the linear to
supercoiled ratio increased with time and the SC: OC ratio remained identical to control
(Thierry et al. 1997). However this view fails to recognize endonucleolytic activity on
linear pDNA also generates degradation products. If endonuclease activity is the primary
route of degradation, the kinetic ratios should all remain similar, owing to the fact that
exonuclease activity would be masked by endonuclease activity. The pharmacokinetics
of this degradation remain to be determined and may serve as a valuable tool in the
understanding of the mechanisms of pDNA degradation observed in the bloodstream.

17
Pharmacokinetics of Liposomal Delivery Vehicles
Although few studies are available on the pharmacokinetics of liposome:pDNA
complexes, liposomal pharmacokinetics alone have been studied extensively with several
reviews published (Hwang et al. 1997; Juliano 1988; Takakura et al. 1996). Liposome
pharmacokinetics have been shown to be dependent upon size (Sato 1986), dose
(Bosworth 1982; Osaka et al. 1996), lipid composition (Gabizon 1988), and charge
(Juliano 1988). In general, liposomes larger than 60 nm in diameter are unable to access
tissues having continuous capillary endothelia, including skeletal, cardiac, and smooth
muscle, lung, skin, subcutaneous tissue, and serous and mucous membranes, and are
limited to uptake in tissues of the reticuloendothelial system (Hwang et al. 1997).
Liposomes larger than 0.5 pm are confined to the vasculature in all tissues.
Pharmacokinetics of Naked Plasmid DNA and Liposome: Plasmid DNA Complexes
After systemic administration of pDNA alone or as liposome:pDNA complexes,
DNA rapidly disappears from the bloodstream. The processes responsible involve
degradation in the blood stream, interaction with plasma proteins, organ distribution, and
uptake by the reticuloendothelial system. The transport of DNA and liposome:pDNA
complexes into organs is roughly a unidirectional system, where distribution back into
the central compartment can be assumed to be negligible (Mahato et al. 1997).
Pharmacokinetics of Naked Plasmid DNA in the Blood after IV Injection
Plasma levels of pDNA may be measured using radiolabeled DNA or agarose gel
analysis (Kawabata et al. 1995; Lew et al. 1995; Mahato et al. 1995; Osaka et al. 1996;
Thierry et al. 1997). Using agarose gel analysis, Thierry and coworkers found that SC
plasmid DNA is not detectable in either murine plasma or cell fractions 1 minute after
injection of naked plasmid DNA in mice (Thierry et al. 1997). OC and L forms have

18
been detected through 30 minutes post-injection by Southern blot analysis (Lew et al.
1995). The half-life of intact (OC or L) plasmid DNA is less than 5 minutes. Degraded
plasmid fragments remain detectable in the blood at 30 minutes post injection. By 60
minutes even degraded plasmid is cleared. This elimination has been shown to be
independent of the DNA sequence (Lew et al. 1995).
When plasmid DNA was administered to mice in the form of liposome:pDNA
complexes, SC DNA was detected in the blood between 1 and 60 minutes after injection
(Thierry et al. 1997). OC DNA degrades with a half-life of approximately 10 to 20
minutes. Uptake of pDNA in blood cells reaches a maximum as early as 1 minute after
injection of liposome:pDNA complexes.
A major problem associated with these studies is that the analysis of samples was
done only qualitatively. No attempt was made to quantitate the amounts and types of
plasmid present in the bloodstream at various times. This information is critical for an
evaluation of the predictive value of pharmacokinetic parameters associated with gene
delivery.
Quantitative analysis of gene delivery has been done using IV injected
radiolabeled pDNA, [32P] or [33P], in mice. In these studies, the half-life of naked pDNA
is approximately 10 minutes (Kawabata et al. 1995; Osaka et al. 1996). The total plasma
radioactivity displays a degradation pattern consistent with a two compartment body
model (Mahato et al. 1995). Total body clearance of naked pDNA is estimated at 102
ml/hr, and plasma AUC is estimated at 0.98 (% of dose*hr/ml). Urinary radioactivity
increases with time, indicating the degradation products are excreted via the kidney.
Similar results were obtained following injection of radiolabeled liposome:pDNA

19
complexes with AUC’s of 0.57 to 0.7 (% dose*hr/ml) and total clearance ranging from
175.8 to 142.7 (ml/hr) (Mahato et al. 1995). Half-life for radiolabeled pDNA:
dimethyldioctadecylammonium bromide: dioleoylphosphatidylethanolamine complexes
was shorter ranging from 4 to 8 minutes (Osaka et al. 1996) suggesting rapid tissue
entrapment of the liposome:pDNA complexes relative to naked plasmid. Twenty-four
hours after injection, blood cell and plasma radioactivity for naked pDNA and
liposome:pDNA complexes were similar (Osaka et al. 1996).
Between these 3 analysis methods (agarose gel, Southern blotting, and
radiolabeling) agarose gel analysis can determine more detailed information on the
degradation of different structures of plasmid, (SC, OC, and L). This method is easy to
apply and can be done under normal conditions without the limitations associated with
radioactivity. The disadvantage of this method is that it is traditionally a semi-
quantitative method. The advantage of the [33P] and [32P] methods is that these are
quantitative methods and are more sensitive than agarose gel analysis. The disadvantages
are that radiolabeling yields OC pDNA and thus, this method gives no information on the
pharmacokinetics of SC pDNA. OC plasmid can also not be differentiated from L
pDNA. The radioactivity is also counted without discriminating the degraded DNA
fragments or the free label. Furthermore, special conditions and precautions are needed
to handle radioactive materials. The difference between [ P] and [ P] is that [ P] has
less personal danger and offers greater ease of handling than [ P] (Song et al. 1997;
Niven et al. 1998).
Overall, when pDNA is injected in mice, SC pDNA has not been detected when
administered as naked pDNA, but is after the injection of liposome:pDNA complexes.

20
After administration as naked pDNA, OC and L pDNA degrades with a half-life of
between 5 and 10 minutes. The half-life of OC or L pDNA after administration of
liposome:pDNA complexes ranges from 4 to 20 minutes. OC pDNA is available for
transcription if taken up by cells. (Adami et al. 1998; Niven et al. 1998) Thus,
administering pDNA in the form of liposome:pDNA complexes may offer a slight
increase in the availability of IV administered pDNA.
Distribution of Plasmid DNA in Tissues after IV Injection
Tissue distribution of pDNA may be measured using radioactivity, Southern
analysis, or whole body autoradiography. Using Southern analysis, pDNA has been
detected in the bone marrow, heart, kidney, liver, lung, spleen, and muscle as early as 1
hour after injection (Lew et al. 1995; Niven et al. 1998). No plasmid was detectable in
the brain, intestine, and ovaries.
Sub-picogram levels may be detected using polymerase chain reaction (PCR).
Using this method, Lew and coworkers showed that at 7 days after IV injection, the range
of residual plasmid was 1 fg/pg in the brain, intestine, and gonads, and was 64 fg/pg in
the marrow, heart, liver, spleen, and muscle (translating to approximately 250-16,000
copies/genome (Lew et al. 1995). By 28 days post-injection, levels of detectable plasmid
had decreased 128 fold. Using PCR, residual plasmid remained detectable 6 months post
injection at 2 to 8 fg/pg genomic DNA and was predominantly in the muscle.
After injection of radiolabeled plasmid, distribution may be measured by isolating
tissues and measuring homogenates in a scintillation counter (Kawabata et al. 1995;
Mahato et al. 1995). Alternatively, the entire carcass may be measured by whole body
sectioning and autoradiography (Osaka et al. 1996; Niven et al. 1998).

21
After injection of radiolabeled naked pDNA, accumulation of radioactivity occurs
initially in the lung, but declines rapidly through 1 minute post-injection (Kawabata et al.
1995). Osaka and coworkers found that by 2 minutes after injection of naked pDNA,
organ distribution is liver>spleen>lung, blood (Osaka et al. 1996). Whereas, Niven and
coworkers found the time to reach maximum levels in the lungs is as long as 5 minutes
versus 2 hours in the liver (Niven et al. 1998). Thus, there appears to be an initial rapid
entrapment and transient accumulation in the lungs with accumulation occurring in the
liver after a short period of time. Plasmid DNA was preferentially recovered in the non-
parenchymal cells in the liver suggesting that the liver is acting in a scavenger role in
uptake (Kawabata et al. 1995).
When compared to naked pDNA, IV injection of liposome:pDNA complexes
shows a higher accumulation of radioactivity in the lung 2 minutes after injection, Osaka
and coworkers showed the major organs exhibit a distribution of
lung>liver>spleen>kidney (Osaka et al. 1996). One hour after injection, a slight rise is
seen in most organs, which is probably related to continuous uptake by the
reticuloendothelial system. By 24 hours after injection of liposome:pDNA complexes,
lung radioactivity dropped approximately 70 fold, with a distribution in major organs of
spleen>liver>lung, kidney.
Conclusions
A complete understanding of the classical pharmacokinetic parameters of gene
delivery is necessary to move genetic agents forward as clinical therapeutics. Problems
include the rapid clearance of naked pDNA and liposome:pDNA complexes without
expression of the gene products, poor target tissue specificity, and degradation in the
plasma. After systemic administration in mice, plasmid DNA is rapidly eliminated from

22
the circulation by extensive uptake by the reticuloendothelial system and degradation by
plasma nucleases. Hepatic uptake is almost identical to liver blood flow suggesting
highly efficient uptake. A complete pharmacokinetic model of all 3 forms of plasmid
DNA (SC, OC, and L) has not been proposed. As the products of the biotechnology
industry begin to move towards more clinical applications, the pharmacokinetic modeling
of gene delivery will likely become an intensely investigated area.

CHAPTER 2
PHARMACOKINETICS OF PLASMID DNA IN ISOLATED RAT PLASMA
Introduction
In vivo delivery of plasmid DNA (pDNA) encoding for therapeutic proteins to
patients via parenteral administration is an attractive means by which to target the gene to
a wide variety of tissues. Early studies revealed that endogenous enzymes present in the
plasma play a role in the clearance of nucleotides from the bloodstream (Chused 1972;
Whaley 1972; Chia 1979; Piva 1998). These early studies have displayed that pDNA
incubated in the presence of 10 % fetal bovine serum shows initial degradation by 15 min
and is completely degraded by 60 min (Piva 1998). Similar results have been displayed
in the presence of 90% human serum (Piva 1998).
Nucleases will convert the native supercoiled (SC) pDNA topoform to the open
circular (OC) and linear (L) forms of the plasmid (Lodish 1995). Changes in topoform
have been associated with alterations in transcriptional activity. The significance of this
change has been the matter of some debate. For example, the OC form of the plasmid
has been shown to express similar levels of chloramphenicol acetyl transferase and
luciferase proteins (Adami et al. 1998; Niven et al. 1998) to 2 to 4 times less (Hirose
1993; Chemg 1999; Ramsey 1999) levels of transcribed luciferase and lac-Z, proteins.
Increases in the amount of supercoiling serves to further increase the percent maximal
transcription (Ramsey 1999). Furthermore, the time required for formation of the
transcription preinitiation complex has been shown to be decreased with a SC template
(Hirose 1993). Degradation to the L form of pDNA is associated with significant losses
23

24
in transcriptional activity (90-100%) (Hirose 1993; Adami et al. 1998; Niven et al. 1998;
Chemg 1999; Ramsey 1999). Differences in transcriptional activity may need to be
accounted for in future pharamcodynamic studies.
Early studies revealed that serum nucleases play a role in the rapid clearance of
genomic DNA from the circulation of injected animals (Gosse et al. 1965). Recent
studies on the pharmacokinetics of pDNA have attempted to use radiolabeled pDNA for
detection (Osaka et al. 1996; Niven et al. 1998). However, the radiolabeling procedure
involves nick translation, thereby eliminating the possibility of maintaining the SC
topoform. Furthermore, this method does not discriminate the degraded pDNA from the
intact plasmid, thus yielding an overestimation of the true half-life of the intact pDNA.
Other studies on the pharmacokinetics of SC and OC pDNA have been only qualitative
citing the presence of pDNA topoforms at various time points (Kawabata et al. 1995;
Osaka et al. 1996; Thierry et al. 1997).
Thierry and coworkers studied the stability of pDNA in the bloodstream of mice
after IV injection (Thierry et al. 1997). Their results indicated that SC plasmid was not
detectable in the plasma or red blood cell fractions 1 min after injection of pDNA. The
true half-life was unable to be calculated using their method due to this rapid degradation.
Kawabata and coworkers found that the SC pDNA was completely converted to the OC
topoform within 5 min when incubated in mouse whole blood (Kawabata et al. 1995).
Little other information on the pharmacokinetics of pDNA is available. The exact
pharmacokinetics underlying this rapid degradative process is not fully understood. To
properly dose and reach the desired therapeutic endpoints, a thorough understanding of
the pharmacokinetics of pDNA is a necessity.

25
It is necessary to study the effects of plasma on pDNA in order to begin
understanding the importance of the degradation of pDNA in the blood and allow a
foundation upon which comparisons of delivery vehicles can be made. Naked pDNA has
been shown to remain in the plasma fraction of blood (Osaka et al. 1996). For these
reasons, we sought to investigate the pharmacokinetic processes underlying the stability
of pDNA in a rat plasma model. We further sought to construct a complete
pharmacokinetic model to describe the degradation of all three topoforms of pDNA in
plasma. This model will allow a prediction of the time course of potential tissue
exposure to the transcriptionally active SC and OC pDNA topoforms.
Methods
Phenol: chloroform: isoamyl alcohol (25: 24: 1, v/v/v), Tris, boric acid, EDTA,
and agarose were purchased from Sigma Chemical Company (St. Louis, MO). Ethidium
bromide (electrophoresis grade) was purchased from Fisher Biotech (Fair Lawn, NJ).
Competent JM109 bacteria (Promega, Madison, WI) were transformed according to the
manufacturers directions with the pGL3 control plasmid (Promega, Madison, WI),
pGeneMax-Luciferase (Gene Therapy Systems, San Francisco, CA) or pGE150 plasmid
(a generous gift of Dr. G. Elliot, Marie Curie Research Institute, The Chart, Oxted,
Surrey, UK). Representative plasmid maps are presented in Figures 2-1, 2-2, and 2-3 for
the pGL3, pGeneMax-Luciferase, and pGE150 plasmids, respectively. Plasmid DNA
was isolated from overnight cultures using the Plasmid Maxi-Prep kit (Quiagen,
Valencia, CA), and was >95% SC by agarose gel analysis.
Blood was isolated from male Sprague-Dawley rats (300-350 g) by cardiac
puncture, and immediately placed in heparinized test tubes (Vacutainer, Becton

26
Figure 2-1. Plasmid map of pGL3 Control.

27
Figure 2-2. Plasmid map of the pGeneMax-Luciferase.

28
Figure 2-3. Plasmid map of pGE 150.

29
Dickinson, Franklin Lakes, NJ) on ice at the times indicated. Blood samples were
centrifuged at 6,000 g for 5 min. For dilution experiments, plasma was diluted to 25 and
50% with PBS or PBS containing 0.1 mM EDTA. To analyze the effects of heat, plasma
samples were incubated at 90°C for 10 min in sealed tubes before assay. Plasma (600 pi)
was removed and placed on ice until assay. Plasma samples were warmed to 37° C in a
water bath and maintained at 37° C for the duration of the experiment. Plasmid DNA (12
pi/ 17 pg) in TE buffer was incubated in the 37° C plasma and 50 pi samples were taken
at various times. Phenol: chloroform: isoamyl alcohol (25: 24: 1, v/v/v) (80 pi) was
immediately added to each sample, vortexed for 5 s at low speed, and placed on ice.
Samples were centrifuged at 20,800 x g for 10 min at room temperature. From the
supernatant, an aliquot of 15 pi was removed, 5 pi of 6 x loading dye (Promega,
Madison, WI) added and placed on ice until loaded on an agarose gel.
Samples were loaded on 0.8% agarose in 0.9 M Tris-Borate and 1 mM EDTA
gels containing 0.001% ethidium bromide. Electrophoresis was carried out at 0.30 V/cm3
for 12 h. The ethidium bromide was excited on a UV light box (Model TFX-35M, Life
Technologies, Grand Island, NY) and the net fluorescence intensity captured at 590 nm
on a Kodak DC 120 digital camera (Eastman Kodak, Rochester, NY). The amounts of
SC, OC, and L pDNA were calculated using Kodak Digital Science ID Image Analysis
Software (version 3.0, Eastman Kodak Company, Rochester NY) with a Lambda Hind III
digest (Promega, Madison WI) and a High DNA Mass Ladder (Life Technologies, Grand
Island, NY) as external standards. Accuracy was 96 to 104% for SC pDNA, 98 to 108%
for OC, and 96 to 113% for L pDNA. Percent coefficient of variation was < 5%, 19%,
and 13% for SC, OC and L forms of the plasmid respectively. Standard curves for all

30
three forms of the plasmid (Figures 2-4, 2-5, and 2-6) were linear between 10 and 250 ng
pDNA bands (R: = 0.9995, 0.9985, and 0.9933 for SC, OC, and L respectively). All
reported concentrations were calculated from bands within the range of the standard
curves. Lower limit of quantitation was 0.5 ng/pl. for all three forms of the plasmid.
Lower limit of detection was 0.25 ng/pl for all three forms of the plasmid. Method
parameters are summarized in Table 2-1. Comparisons of the relative fluorescence of SC
pDNA versus OC and L pDNA were made by analyzing equivalent amounts as described
above and comparing the resulting fluorescence. It was found that on a weight to weight
ratio, SC pDNA was only 59% as fluorescent relative to L pDNA by agarose gel analysis.
To correct for this difference, SC pDNA amounts were multiplied by 1.7 prior to
analysis. This difference has been reported previously and is likely due to the relative
inaccessibility of ethidium bromide to the SC topology (Cantor 1980). Percent recovery
using the phenol: chloroform: isoamyl alcohol method was found not to be dependent on
topoform. Recovery was 90 (± 6) % for SC and 86 (± 13) % for L pDNA. Comparisons
of the relative fluorescence of SC pDNA versus L pDNA were made by digesting SC
pDNA with the Hind III restriction enzyme (Promega, Madison, WI) which has a single
recognition site in the plasmid. Equivalent amounts of L and SC pDNA were then loaded
on agarose gels as described above and the relative fluorescence compared. Percent
recovery was calculated by comparing phenol: chloroform: isoamyl alcohol (25: 24: 1,
v/v/v) extracted versus non-extracted known amounts and analyzing on agarose gels as
described above.

31
A260 DNA equivalents
Figure 2-4. Standard curve for supercoiled pDNA. Abcissa represents total ng estimated
by UV absorbance at 260 nm. Ordinate represents ng calculated by the KDS ID
software. Error bars represent ±1 standard deviation.

Calculated
32
Figure 2-5. Standard curve for OC pDNA. Abcissa represents total ng estimated by UV
absorbance at 260 nm. Ordinate represents ng calculated by the KDS ID software. Error
bars represent ±1 standard deviation.

Calculated
33
Figure 2-6. Standard curve for Linear pDNA. Abcissa represents total ng estimated by
UV absorbance at 260 nm. Ordinate represents ng calculated by the KDS ID software.
Error bars represent ±1 standard deviation.

34
Table 2-1. Method parameters for pDNA analysis.
Accuracy
Supercoiled: 94-101 %
Open Circular: 98-108 %
Linear: 96-113 %
Precision
Supercoled: <5 %
Open Cicular: <19 %
Linear: <13 %
Lower Limit of Quantitation
0.5 ng/pl
Lower Limit of Detection
0.25 ng/pl
Recovery from Plasma
Supercoiled: 90 (±6) %
Linear: 86 (±13) %

35
Theoretical
The degradation of SC pDNA was assumed to follow pseudo first-order kinetics.
The model used is diagrammed in Figure 2-7. In this model, pDNA degradation is
considered to be a unidirectional process. The degradation of L pDNA is considered to
yield fragments of heterogeneous lengths, thus these products were not included in the
fitted model. No elimination from any of the compartments is assumed to occur through
routes other than degradation to the following topoform.
Based on this model the following differential equations were derived to describe
the process:
dSC
dt
dOC
dt
= —ks ■ SC
= k-SC-k-OC
— = k -OC-k.-L
dt
The amounts of supercoiled, open circular and linear pDNA were then fit to the
integrated form of the equations:
SC = SC0-e~k,t
OC = k,SC„-(
k = K -k' SCf,
■ e~k°' +■
")
Where SC, OC, and L are the amounts of supercoiled, open circular, and linear pDNA
present at time=t, respectively. SC0 is the amount of supercoiled pDNA present at time
(t)=0. The constants ko ,ks, and k] represent the rate constants for the degradation of SC,
OC, and L pDNA respectively. The constants represent the activity of all enzymes acting
in the degradation process. Non-linear curve fitting and goodness of fit, model selection

36
criteria (MSC) assessment was carried out using Scientist (version 4.0, Micromath, Salt
Lake City, UT) (MicroMath 1995). Area under the plasma concentration time curve
(AUC) was calculated using trapezoidal rule. Area under the terminal portion of the
plasma concentration time curve, AUCtemi, was calculated by integration using the
equation:
AUCterm = c-f-
Where Ciast is the last concentration point measured and k is the terminal elimination rate
constant. Clearance (Cl) was calculated from the volume (V) of rat plasma (7.8 ml) and
the terminal elimination rate constant (k) using the equation (Davies 1993):
Cl = V-k
Statistical analysis was performed using SAS (The SAS Institute, Cary, NC).
Results
For quantitative purposes, the relative fluorescence of SC pDNA was compared to
that of OC and L pDNA. It was found that on a weight to weight ratio, SC pDNA was
only 59% as fluorescent relative to L pDNA by agarose gel analysis. To correct for this
difference, SC pDNA amounts were multiplied by 1.7 prior to analysis. This difference
has been reported previously, and is likely due to the relative inaccessibility of ethidium
bromide to the SC topology (Cantor 1980). Percent recovery using the phenol:
chloroform: isoamyl alcohol method was found not to be dependent on topoform.
Recovery was 90 (±6) % for SC and 86 (±13) % for L pDNA.

37
SC
Linear
ki
o
Figure 2-7. Pharmacokinetic model of plasmid DNA degradation in rat plasma. The
model is considered to be a unidirectional process. SC, OC, and L represent the amounts
of supercoiled, open circular, and linear plasmid, respectively, in each compartment. The
rate constants ks, ko, and ki represent the degradation constants for supercoiled, open
circular, and linear plasmid, respectively.

38
Figure 2-8 displays a representative gel in which the degradation of SC pDNA
and the appearance of OC and L topoforms of plasmid is observed. In addition, the
degradation products of L pDNA are visible as a light smear running below the band at
60 min. Under the conditions used in this experiment, limit of quantification was 0.5
ng/pl using the Lambda Hind III size standard. Plasmid amounts were calculated from
agarose gel analysis using Kodak Digital Science ID image analysis software (Eastman
Kodak, Rochester, NY) as described in the methods section. The observed and predicted
values, based on the model displayed in Figure 2-8, are plotted in Figure 2-9. Plasmid
concentrations were well described the model, MSC-3.0. Pharmacokinetic parameters
calculated based on the model are summarized in Table 2-2. SC pDNA degraded rapidly
in the plasma with a half-life of 1.2 (± 0.1) min. OC plasmid however was fairly stable,
degrading with a half-life of 21 (± 1) min. L plasmid degraded more rapidly than the OC
topoform but was fairly stable, in comparison to the SC plasmid degrading with a half-
life of 11 (± 2) min. OC AUC was nearly 17 times larger than SC, and 2.3 times larger
than L pDNA (Table 2-3).
No kinetics suggestive of enzyme saturation were observed under the
experimental conditions tested. However, to ensure that saturation of plasma nucleases
was not resulting in artificially low rate constant values, we analyzed the rate constants
produced in dilute plasma (dilution was chosen as decreasing the dose of pDNA quickly
results in a loss of sensitivity and sample sizes too large for loading). If saturation of

39
1
2 3 4 5 6 7
8 9 10 11 12
Figure 2-8. Agarose gel analysis of pDNA degradation in isolated rat plasma. Lane 1;
size standard, lane 2; 30 sec, lane 3; 1 min, lane 4; 2 min, lane 5; 3 min, lane 6; 5 min,
lane 7; 10 min, lane 8; 20 min, lane 9; 30 min, lane 10; 45 min, lane 11; 60 min, lane 12
80 min.

(|ri/6u) VNQd
40
time (min)
Figure 2-9. Experimental and fitted data based on the pharmacokinetic model described
in the text. Data points represent actual experimental data. Lines represent values
predicted by the model. Data represents mean of n=3 ± 1 standard deviation. Key: B
supercoiled, • open circular, ▲ linear.

41
Table 2-2. Pharmacokinetic parameters for pDNA in isolated rat plasma
Topoform
Rate
constant
Value (min')
Standard
Deviation
Half-life (min)
Supercoiled
ks
0.6
0.03
1.2 (±0.1)
Open circular
k0
0.03
0.002
21 (±1)
Linear
kt
0.06
0.008
11 (±2)
Data represent the
itted values of n=6 ± 1 standard deviation.

42
Table 2-3. Noncompartmental parameters for pDNA after incubation in isolated plasma.
Topoform
AUC (ng/pl*min)
Clearance
(pl/min)
Supercoiled
18
360 (± 9)
Open circular
310
23 (± 1)
Linear
130
47 (± 5)
Data represent n=6 ± standard deviation. AUC was calculated from the model fitted
values using trapezoidal rule as described in the methods section. Clearance was
calculated from the fitted rate constants and volume of rat plasma as described in the
Methods section.

43
plasma nucleases was occurring, we expected that the rate constants in dilute plasma
should deviate from a linear relationship negatively. Thus we tested the kinetics of
pDNA degradation in 25% and 50% plasma. As displayed in Figure 2-10, no deviation
was observed in the degradation of SC and OC pDNA.
To further investigate the mechanism responsible for the observed degradation,
and further validate our assay (to ensure the assay was not causing degradation itself) we
studied the degradation of pDNA in PBS diluted 25% heated plasma (90°C for 10 min)
and PBS containing 0.1 mM EDTA. No degradation of SC pDNA was observed in either
case through 1 hr (Figure 2-11). The degradation sensitivity to heat and EDTA provides
evidence that the degradation observed in the assay is due to enzymatic processes.
We next sought to determine if the degradation observed in the previous
experiments was dependent upon pDNA sequence. We, therefore, utilized the same
model diagrammed above and replaced the pGL3 plasmid with the pGE150 plasmid.
Unlike the pGL3 plasmid, which encodes for the luciferase protein and has an SV40
promoter, this plasmid encodes for the green fluorescent protein and includes a CMV
promoter. If the degradation of pDNA in the plasma was sequence dependent, we
expected the degradation rate constants observed to differ from those observed in the
previous experiment. A comparison of plasma concentrations of OC and L pDNA is
presented in Figures 2-12 and 2-13. The resulting rate constants are presented in Table 4.
Again the model diagrammed in Figure 2-7 described the data (model selection
criteria = 3.1, R2=0.96, 0.99, and 0.92 for SC, OC, and L respectively). To determine if

44
0.7
0.6
0.5
c 0.4
I 0.3
0.2
0.1
0
0
y = 0.6494X - 0.0311
% plasma
+ Ks
â–  Ko
1.2
Figure 2-10. Analysis of rate constants in dilute plasma. Degradation rate constants were
modeled in PBS diluted rat plasma. Rate constants represent the fitted values of n=6 rats/
time point. Key: ♦ks in dilute plasma, «ko in dilute plasma. The value of k] is not
reported due to the prolonged stability of linear plasmid in dilute plasma.

45
A B
Std 0.5m lm 2m 3m 5m 10m 15m 20m 30m 45m lhr Std 0.5m lm 2m 5m 10m 20m 30m 45m lhr
Figure 2-11. Degradation of supercoiled pDNA in 25% rat plasma after (A) incubating
the plasma at 90°C for 10 min (B) the addition of 0.1 mM EDTA.

46
these rates were significantly different from those obtained using the pGL3 plasmid a
statistical analysis was carried out using a 2-tailed equal variance student’s t-test. The
resulting parameters (ko, and k|) were not significantly different when judged at the
p<0.05 criteria. These results suggest that pDNA sequence is not a major factor involved
in the overall degradation of pDNA by plasma nucleases.
Conclusions
Previous reports on the pharmacokinetics of pDNA have only been qualitative, or
involved radiolabeling. These studies indicated that pDNA degrades within 5 minutes in
vitro or after IV injection (Kawabata et al. 1995; Thierry et al. 1997). In this study, we
sought to quantitatively model the pharmacokinetics underlying the stability of pDNA in
the plasma. The results revealed that SC pDNA degrades in the plasma with a half-life of
1 min. OC pDNA is more stable than the SC topoform degrading with a half-life of 20
min. L pDNA is degraded more rapidly than the OC topoform. This latter shortened
stability is likely due to the accessibility of various nucleases present in the plasma to the
L pDNA. OC plasmid must be nicked by endonucleases on each sister strand in the same
location to generate L pDNA. However L pDNA would be accessible to both
endonucleases and exonucleases, thus degrading more rapidly. The model and equations
presented successfully described the degradation of pDNA in the plasma.
Investigations by Thierry and coworkers suggested the main nuclease activity was
endonucleolytic based on the finding that the L: SC ratio increased over time and the SC:
OC ratio remained identical to control (Thierry et al. 1997). However this view fails to

pDNA (ng/ul)
47
100
0 20 40 60 80
time (min)
Figure 2-12. Comparison of concentrations of OC pDNA using (♦) pGE150
concentrations of OC pDNA using (â– ) pGL3 in isolated rat plasma. Data represents
mean of n=3 ±1 standard deviation.

pDNA (ng/ul)
48
0 20 40 60 80
time (min)
Figure 2-13. Comparison of concentrations of L pDNA using (♦) pGE150 versus
concentrations of L pDNA using (â– ) pGL3 in isolated rat plasma. Data represents mean
of n=3 ±1 standard deviation.

49
Table 2-4. Pharmacokinetic parameters for pDNA after incubating the pGE150 plasmid
in isolated rat plasma.
Standard
Topoform
Rate constant
Value (min ')
Deviation
Half-life (min)
Open Circular
k0
0.04
0.007
21 (±1)
Linear
ki
0.06
0.007
11 (±1)
Data represent the fitted values of n=6 rats.

50
recognize endonucleolytic activity on L pDNA also generates degradation products. If
endouclease activity is the primary route of degradation, the kinetic ratios should all
remain similar or decrease, owing to the fact that both exonucleases and endonucleases
are active on the L pDNA and are thus both responsible for the observed degradation.
Our results suggest that L pDNA has faster kinetics. This can be explained by
endonuclease activity generating more free ends for degradation by exonucleases,
exonucleases are more active than endonucleases, or that topoform influences the binding
of these enzymes and thus influences reaction rate. Thus the main nuclease activity
responsible for the observed kinetics remains to be answered.
Area under the curve analysis revealed that tissues would be exposed to the OC
topoform predominantly after injection of naked pDNA (Table 2-2). Blood flow through
any individual organ becomes the limiting factor in its ability to uptake a drug, which is
highly metabolized in the plasma. When compared to hepatic plasma flow in the rat
(8.14 ml/min), clearance values for the degradation of SC plasmid (4.6 ml/min) suggest
that metabolism in the bloodstream is a major pathway by which in vivo clearance of SC
pDNA can occur (Davies 1993). However, given that the clearance by degradation in the
plasma is less than the liver blood flow, it also suggests that the liver possess a perfusion
rate sufficient for uptake of SC pDNA after IV injection. This parallels the findings of
Kawabata and coworkers who observed that naked plasmid was cleared more rapidly
from the circulation after IV injection than after in vitro incubation in whole blood
(Kawabata et al. 1995). Lung, kidney, and spleen have also been shown to take up
detectable amounts of plasmid after IV injection (Kawabata et al. 1995; Osaka et al.

51
1996). Our model establishes not only that tissue uptake of plasmid is possible, but also
that tissue uptake of the non-nicked SC topoform is possible after IV injection.
In summary this study presents a pharmacokinetic model describing the
degradation of pDNA in rat plasma. A pharmacokinetic model is presented that can be of
use in the future as gene therapy moves toward clinical trials. Using the derived model,
we are able to conclude that naked SC pDNA degrades in rat plasma with a half-life of
1.2 (± 0.05) min, OC with a half-life of 21 (± 1) min, and L pDNA with a half-life of 11
(± 2) min.

CHAPTER 3
PHARMACOKINETICS OF PLASMID DNA AFTER IV BOLUS ADMINISTRATION
IN THE RAT
Introduction
Naked pDNA is being used successfully in gene delivery by administration IM or
SQ, (Haensler et al. 1999; Noll et al. 1999; Osorio et al. 1999; Rizzuto et al. 1999) and
after IV injection (Wang et al. 1995; Budker et al. 1998; Song et al. 1998; Liu 1999;
Zhang et al. 1999) in rats and mice. The success in these studies indicates that gene
therapy is an attractive means by which to achieve therapeutic response. Thus, a
thorough understanding of the pharmacokinetics of naked pDNA is an important area to
be considered in order to move towards use in clinical trials
The pharmacokinetics of pDNA after IV bolus administration have been
investigated using radiolabeling with linearized [33P] pDNA (Osaka et al. 1996). These
investigations have led to the conclusion that the half-life of the pDNA radiolabel is 7 to
12 min after IV bolus administration of naked pDNA in mice. However, this analysis
offers no information on the other functional forms of the plasmid; supercoiled (SC),
open circular (OC), or linear (L), nor does it discriminate the free label. Other studies
have qualitatively revealed that the SC topoform of pDNA is not detectable as early as
one minute post IV injection in mice (Lew et al. 1995; Thierry et al. 1997). The OC form
of the plasmid has a half-life estimated in these studies to be in the range of 10 to 20
minutes (Thierry et al. 1997).
52

53
The purpose of this investigation was to model the pharmacokinetics of naked
pDNA in a topoform specific manner after IV bolus administration in the rat. We further
sought to determine if the observed pharmacokinetics were affected by changes in
plasmid sequence. These results were then compared to pDNA degradation in isolated
plasma in order to determine the relative importance of plasma nucleases in the
pharmacokinetics of pDNA.
Methods
Animals (male Sprague-Dawley rats 300-350 g) were purchased from Charles
River Laboratories (Wilmington, MA). Animals were housed in the University of Florida
Animal Resources Unit prior to all experiments and were given food and water ad
libitum. Animals were anesthetized by intraperitoneal injection with 0.5 ml of a cocktail
containing 13 mg/kg Xylazine, 2.15 mg/kg Acepromazine (The Butler Co., Columbus,
OH), and 66 mg/kg Ketamine (Fort Dodge Animal Health, Fort Dodge Iowa).
Phenol: chloroform: isoamyl alcohol (25: 24: 1, v/v/v), chloroform, Tris, boric
acid, EDTA, and agarose were purchased from Sigma Chemical Company (St. Louis,
MO). Ethidium bromide (electrophoresis grade) was purchased from Fisher Biotech
(Fair Lawn, NJ). Competent JM109 bacteria (Promega, Madison, WI) were transformed
according to the manufacturer’s directions with the pGL3 control plasmid (Promega,
Madison, WI) or pGeneMax plasmid (Gene Therapy Systems, San Francisco, CA).
Plasmid DNA was isolated from overnight cultures using alkaline lysis and
ultracentrifugation with a CsCl gradient (Katz 1977). Isolated pDNA was resuspended in
phosphate buffered saline. Plasmid was >90% SC by agarose gel analysis.
To facilitate blood sampling, male Sprague-Dawley rats (300-350g) were
anesthetized and the jugular vein was exposed via an incision, isolated, ligated, and

54
nicked with ophthalmic scissors. A sterile silatstic (0.640 cm internal diameter by 0.12
cm outer diameter, 10 cm in length) filled with sterile saline was threaded 30-40 mm into
the jugular vein and positioned just distal to the entrance to the right atrium and secured
by 6.0 silk sutures Figure 3-1). For injections, the femoral vein was isolated, and pDNA
was injected into the femoral vein using a 27-gauge needle (Figure 3-2). Isolated blood
samples (approx. 300 pi) were drawn through the jugular vein cannula and immediately
placed in test tubes containing 0.57 ml of 0.34 M EDTA (Vacutainer, Becton Dickinson,
Franklin Lakes, NJ) on ice at the times indicated. This concentration of EDTA has
previously been shown to inhibit the degradation of pDNA in isolated rat plasma (Houk
1999).
To isolate pDNA from whole blood samples, 250 pi of blood was liquid/ liquid
extracted with 250 pi of phenol: chloroform: isoamyl alcohol (25: 24: 1, v/v/v), vortexed
for 5 s at low speed, and centrifuged at 20,800 x g for 10 min at room temperature. The
aqueous phase was removed and stored at -20°C until analysis.
Samples were loaded on 0.8% agarose in 0.9 M Tris-borate, 1 mM EDTA gels
containing 0.001% ethidium bromide. Electrophoresis was carried out at 0.30 V/cm3 for
12h. The ethidium bromide was excited on a UV light box (Model TFX-35M, Life
Technologies, Grand Island, NY) and the net fluorescence intensity captured at 590 nm
on a Kodak DC 120 digital camera (Eastman Kodak, Rochester, NY). The amounts of
SC, OC, and L pDNA were calculated using Kodak Digital Science ID Image Analysis
Software (version 3.0, Eastman Kodak Company, Rochester NY) with a Lambda Hind III
digest (Promega, Madison WI) and a High DNA Mass Ladder (Life Technologies, Grand

55
Figure 3-1. Photograph of the jugular cannula placement used for blood sampling.

56
Figure 3-2. Photograph of the femoral vein isolation and injection procedure used for IV
bolus administration.

57
Island, NY) as external standards. Accuracy was 96 to 104% for SC pDNA, 98 to 108%
for OC, and 96 to 113% for L pDNA. Percent coefficient of variation was < 5%, 19%,
and 13% for SC, OC and L forms of the plasmid respectively. Standard curves for all
three forms of the plasmid (Figures 2-1, 2-2, and 2-3) were linear between 10 and 250 ng
pDNA bands (R2 = 0.9995, 0.9985, and 0.9933 for SC, OC, and L, respectively). All
reported concentrations were calculated from bands within the range of the standard
curves. Lower limit of quantitation was 0.5 ng/pl for all three forms of the plasmid.
Lower limit of detection was 0.25 ng/pl for all three forms of the plasmid. Method
parameters are summarized in Table 2-1. Comparisons of the relative fluorescence of SC
pDNA versus OC and L pDNA were made by analyzing equivalent amounts as described
above and comparing the resulting fluorescence. It was found that on a weight to weight
ratio, SC pDNA was only 59% as fluorescent relative to L pDNA by agarose gel analysis.
To correct for this difference, SC pDNA amounts were multiplied by 1.7 prior to
analysis. This difference has been reported previously, and is likely due to the relative
inaccessibility of ethidium bromide to the SC topology (Cantor 1980). Percent recovery
using the phenol: chloroform: isoamyl alcohol method was found not to be dependent on
topoform. Recovery was 90 (± 6) % for SC and 86 (± 13) % for L pDNA.
Theoretical
The degradation of SC pDNA was assumed to follow pseudo first-order kinetics.
The model used is diagrammed in Figure 2-3. In this model, pDNA degradation is
considered to be a unidirectional process. The degradation of L pDNA is considered to
yield fragments of heterogeneous lengths, thus these products were not included in the

58
fitted model. No elimination from any of the compartments is assumed to occur through
routes other than degradation to the following topoform.
Based on this model the following differential equations were derived to describe
the process:
— = -ks-SC
dt
(^ = k-SC-k0OC
dt
— = kn OC-k, ■ L
dt 0 1
The amounts of supercoiled, open circular and linear pDNA were then fit to the
integrated form of the equations:
SC=SC0e~k‘‘
OC = ks-SC0-(^_j-.e*
L-k0-k.- SC„ • (-
â– ')
-u
•e~k°'1 +
i
(k0-ks )(k,-ks)
e ks '1 +
i
(k0-k, )(k$-k¡)
Where SC, OC, and L are the amounts of supercoiled, open circular, and linear
pDNA present at time=t, respectively. SCo is the amount of supercoiled pDNA present at
time (t)=0. The constants ks ,ko, and k] represent the rate constants for the degradation of
supercoiled, open circular, and linear pDNA, respectively. The constants represent the
activity of all enzymes acting in the degradation process. Non-linear curve fitting and
statistical analysis was carried out using Scientist (version 4.0, Micromath, Salt Lake

59
Figure 3-3. A representative gel from which plasmid amounts were quantified as
described in the methods section. Lane 1: size standard, lane 2: 1 min, lane 3: 2 min, lane
4: 3.5 min, lane 5: 5 min, lane 6: 10 min, lane 7: 20 min, lane 8: 30 min, lane 9: 45 min,
lane 10: 60 min.

60
City, UT). Noncompartmental pharmacokinetic analysis was carried out using standard
parameters (Gibaldi 1982).
Results
SC pDNA was not detected as early as 30 seconds post-injection. The OC and L
forms of the pDNA remained detectable through 30 minutes post-injection of the 500 pg
dose. An agarose gel analysis of the isolated samples is presented in Figure 3-3.
An important parameter to be considered is the initial concentrations achieved
after IV administration in comparison to the initial concentrations in vitro. The initial
concentrations of SC pDNA in the in vitro experiments (i.e. the time=0 concentration)
were 10 (± 0.3) ng/pl. After IV bolus administration, the initial extrapolated SC pDNA
concentrations were 17 (± 5) ng/pl. Thus, we concluded that these concentrations were
within a range relevant for comparison. The observed and fitted concentrations of OC
and L pDNA are presented in Figure 3-4. The model again adequately described the
data, model selection criteria=4.42. Pharmacokinetic parameters calculated based upon
the model are presented in Table 3-1.
A comparison of the in vitro and in vivo concentrations of OC and L pDNA are
presented in Figures 3-5 and 3-6, respectively. Calculated pharmacokinetic parameters
are presented in Table 3-2. OC pDNA half-life was markedly shorter after IV bolus
administration than after incubation in isolated plasma, 5.3 (±1.4) versus 21 (±1) min. L
pDNA removal was also more rapid after IV bolus administration, 1.9 (+0.8) versus 11
(±2) min after incubation in isolated plasma.
In order to further investigate the importance of plasmid sequence on the observed
pharmacokinetics, we injected the pGeneMax-Luciferase, and pGE150 plasmids by IV

61
bolus administration at equivalent dose (500 pg). Concentrations of OC and L pDNA in
the bloodstream are presented in Figures 3-7 and 3-8 respectively. The fitted elimination
rate constants for OC and L pDNA were compared by 2-way ANOVA. The results are
displayed in Table 3-3. There were no significant differences between the terminal rate
constants of any of the 3 plasmids by 2-way ANOVA when judged at the p<0.05 criteria.
Conclusions
DNase I is a well characterized enzyme in human plasma present at
concentrations averaging 26.1 (±9.2) ng/ml in the sera of normal humans (Chitrabamrung
1981). Traditionally, the presence of this enzyme has led to the conclusion that pDNA
administered IV is degraded rapidly (Gosse et al. 1965; Chused 1972). This has led to
the current view of gene delivery, where protection from plasma nucleases is a major
goal of delivery systems. The results of this study demonstrate that although the half-life
of SC and OC pDNA is remarkably short, degradation alone was not enough to explain
the rapid disappearance of pDNA from the circulation observed in vivo. After IV bolus
the rate of degradation of SC pDNA was greater than 7 times faster than in isolated
plasma (Houk 1999).
Chused and coworkers (Chused 1972) also suggested that nuclease activity was
not enough to explain the rapid clearance of KB cell DNA from the circulation in mice.
In this study, only 2 to 3 % of the radioactivity was hydrolyzed to trichloroacetic acid
(TCA) soluble fragments in 30 min, which was several half-lives longer than in the
circulation. Tsumita and Iwanga (Tsumita and Iwanga 1963) also found that less than 5
% of the total radioactivity was found in the TCA soluble fraction after 4.5 hours in
mouse serum.

62
O 10 20 30 40 50
time (min)
Figure 3-4. Experimental and Fitted data based on the pharmacokinetic model described
in the text. Data points represent actual experimental data. Lines represent values
predicted by the model. Data represents mean of n=6 ± 1 standard devaition. Key: •
open circular, â–² linear.

pDNA (ng/ul)
63
20
Figure 3-5. Concentrations of OC pGL3 after â– : IV bolus administration of a 500 pg
dose of SC pGL3, and ♦: Incubation of SC pGL3 in isolated plasma at 37°C.
80

pDNA (ng/ul)
64
80
Figure 3-6. Concentrations of L pGL3 after â– : IV bolus administration of a 500 pg dose
of SC pGL3, and ♦: Incubation of SC pGL3 in isolated plasma at 37°C.

65
Table 3-1. Pharmacokinetic parameters calculated after 500 pg dose of SC pDNA.
Topoform
Rate
Value
Standard
Half-life
constant
(min'1)
Deviation
(min)
Supercoiled
ks
3.4
0.4
0.2 (± 0.03)
Open circular
k0
0.14
0.04
5.3 (± 1.4)
Linear
ki
0.41
0.18
1.9 (±0.8)
Parameters represent averages of n=6 rats.

66
Table 3-2. Comparison of in vivo and in vitro pharmacokinetic parameters for pDNA.
Topoform
Terminal Half-life
(min)
AUCoo (ng/pl*min)
Cl/f (pl/min)
In vitro
In vivo
In vitro In vivo
In vitro
In vivo
Supercoiled
1.2 (±0.1)
?
17(± 5)
N/A
360 (± 9)
N/A
Open Circular
21 (±1)
5.3 (±1.4)
280
(±150)
128 (± 52)
23 (± 1)
4800
(±2000)
Linear
11 (± 2)
1.9 (±0.8)
103 (± 47)
49 (± 28)
47 (±5)
11000
(±5000)
Parameters represent averages of n=3 (± 1 standard deviation).

pDNA (ng/ul)
67
pCMV-Luc
pGE150
pGL3
15
Figure 3-7. Concentrations of OC pDNA in the bloodstream after IV bolus
administration of 500 pg of pCMV-Luc, pGE150, or pGL3. Data represents mean of n=3
± 1 standard deviation.

pDNA (ng/ul)
68
10
0
10 20
time (min)
30
Figure 3-8. Concentrations of L pDNA in the bloodstream after IV bolus administration
of 500 (ig of pCMV-Luc, pGE150, or pGL3. Data represents mean of n=3 ± 1 standard
deviation.

69
Table 3-3. Comparison of OC and L pDNA parameters after administration of SC pGL3,
pGE150, and pGeneMax.
Plasmid
Parameter
OC Value
L Value
pGL3
AUCoo
(ng/|il*min)
120(±50)
52 (±25)
Cmax (ng/p.1)
13 (±4)
3.2 (±1.0)
pGE150
AUCoo
(ng/pl*min)
160(±30)
55(±12)
Cmax (n§/M"l)
14 (±3)
3.3 (±0.9)
pGeneMax
AUCoo
(ng/pl*min)
121(±25)
59 (±3)
Cmax (ng/pl)
12 (±5)
3.7 (±0.3)
Parameters represent averages of n=3 (± 1 standard deviation).

70
Alternatively, Gosse and coworkers suggested a major role for nucleases in the
initial degradation of DNA after IV administration in rabbits and mice (Gosse et al.
1965). This finding was based upon the proportionality between the initial rate of
depolymerization and the plasma DNase activity level. Also, a rapid decrease in
viscosity of isolated blood was discovered indicating a depolymerization of DNA.
Finally, a markedly slower disappearance of DNA-methyl green complex (a non-specific
DNase inhibitor) than after native DNA.
The reason for this disparity in results deserves further investigation. Gosse
utilized much higher doses of pDNA than Chused and coworkers in their investigations,
200 pg versus 5 pg pDNA in Chused and coworkers ’s investigations. This disparity
may be due to saturation of a scavenger receptor, allowing nuclease activity to become
increasingly important. The effect of increasing dose on the clearance of DNA deserves
further investigation.
The results presented in the present study indicate that SC pDNA was
undetectable after IV bolus administration, whereas SC pDNA was readily detectable in
isolated plasma, and remained detectable through 3 min of incubation. Similar results
were seen for the OC and L forms of the plasmid. The half-lives of OC and L pDNA
decreased from 21 (±1) to 5.3 (+1.4) and 11 (±2) to 1.9 (±0.8) min, respectively. Thus
indicating that nuclease activity alone is not sufficient to describe the rapid clearance of
pDNA from the bloodstream in rats. The observed kinetics were found not to be
dependent upon plasmid sequence.

CHAPTER 4
DOSE DEPENDENCY OF PLASMID DNA PHARMACOKINETICS
Introduction
The studies presented in Chapters 2 and 3 have shown that degradation in the
plasma alone was not sufficient to describe the pharmacokinetics of pDNA. After IV
bolus administration SC pDNA was undetectable as early as 30 sec. This was in contrast
to isolated plasma when SC pDNA was detectable 3 minutes after the start of incubation
in isolated plasma. OC and L pDNA terminal half-life also decreased from 21 (±1) to 5.3
(±1.4) and 11 (±2) to 1.9 (±0.8) min, respectively.
Other investigators have suggested variable importance of plasma nucleases in the
degradation of genomic DNA after IV bolus administration. For example, Chused and
coworkers (Chused 1972), Whaley and Webb (Whaley 1972), and Tsumita and Iwanaga
(Tsumita 1963) all suggested a minimal role for plasma nucleases in the clearance of
DNA. This was based upon the observed fragmentation rate of genomic DNA in isolated
plasma versus the fragmentation rate after IV bolus administration, and the diffuse high
level of distribution of the DNA to tissues immediately after administration. This finding
was accompanied by the suggestion of extensive uptake of intact DNA molecules by the
reticuloendothelial system.
Alternatively, Gosse and coworkers (Gosse et al. 1965) found that “the plasma
DNases play a fundamental and probably exclusive role in the initial degradation of
DNA”. This was based upon 3 observations. First was a rapid decrease in viscosity of
71

72
the blood within 3 minutes after administration. Second, this was based upon the
proportionality between the initial rate of degradation and the DNase activity level.
Third, this was also based upon the markedly slower disappearance of the DNA-methyl
green complex (a non-specific DNase inhibitor).
The disparity between the results presented here and the previous studies deserves
further investigation. Chused and coworkers, and Whaley and Webb, utilized smaller
doses of DNA in their experiments versus Gosse and coworkers (5 versus 200 pg/
mouse). If this large dose had temporarily saturated an alternative clearance mechanism,
this would increase the observed importance of nucleases. Thus, nonlinear processes
may provide an explanation for the observed disparity.
Nonlinear clearance of pDNA has previously been suggested using
pharmacokinetic analysis of outflow patterns from rat perfused liver studies with
radiolabeled OC pDNA(Yoshida 1996). In this study, Vd increased and extraction ratio
decreased as perfusion dose was increased from 1.33 to 13.3 pg/liver.
The purpose of this investigation was to model the pharmacokinetics of increasing
doses of naked pDNA in a topoform specific manner after IV bolus administration in the
rat. This information may provide an explanation for the disparity between the results
presented here and in previous studies. Furthermore, we sought to determine the
metabolite (OC and L) pharmacokinetics independently, by direct injection of each of the
metabolites. This information will provide a basis upon the percent conversion of the SC
to the OC form and the OC form to the L form of the plasmid.

73
Methods
Animals (male Sprague-Dawley rats 300-350 g) were purchased from Charles
River Laboratories (Wilmington, MA). Animals were housed in the University of Florida
Animal Resources Unit prior to all experiments and were given food and water ad
libitum. Animals were anesthetized by intraperitoneal injection with 0.5 ml of a cocktail
containing 13 mg/kg Xylazine, 2.15 mg/kg Acepromazine (The Butler Co., Columbus,
OH), and 66 mg/kg Ketamine (Fort Dodge Animal Health, Fort Dodge Iowa).
Phenol: chloroform: isoamyl alcohol (25: 24: 1, v/v/v), chloroform, Tris, boric
acid, EDTA, and agarose were purchased from Sigma Chemical Company (St. Louis,
MO). Ethidium bromide (electrophoresis grade) was purchased from Fisher Biotech
(Fair Lawn, NJ). Competent JM109 bacteria (Promega, Madison, WI) were transformed
according to the manufacturers directions with the pGL3 control plasmid (Promega,
Madison, WI) or pGeneMax plasmid (Gene Therapy Systems, San Francisco, CA).
Plasmid DNA was isolated from overnight cultures using alkaline lysis and
ultracentrifugation with a CsCl gradient (Katz 1977). Isolated pDNA was resuspended in
phosphate buffered saline. Plasmid was >90% SC by agarose gel analysis.
OC pDNA was produced by incubation of the SC pDNA, in phosphate buffered
saline, at 70°C for 16h. This procedure resulted in >90% OC plasmid (Figure 4-1). UV
absorbance at 260 nm and the A260/A280 ratio of the pDNA solution did not change
after this treatment (Figure 4-2).
L pDNA was produced by digestion with BamHI restriction enzyme (Promega,
Madison, WI) in separate reaction mixtures containing 173 pi of DI H20, 27 pi lOx
Buffer (Promega, Madison, WI) 56 pi (100 pg) pGL3, and 10 pi of BamHI (10 U/pl).

74
The reaction mixture was incubated at 37°C for 3 h. Plasmid was then isolated from the
reaction mixture by extraction with 1 volume of phenol: chloroform: isoamyl alcohol (25:
24: 1), followed by extraction with 1 volume of chloroform. Plasmid was then
concentrated by precipitation with 0.3 M Na Acetate, and 1 volume of isopropanol,
followed by centrifugation at 13K g for 30 min at 4°C, and resuspended in 50 pi of
phosphate buffered saline. This method routinely produced >90% L pDNA (Figure 4-3).
Concentration of pDNA was measured by monitoring UV absorbance at 260 nm, purity
was measured by A260/A280 ratio. A resulting purity of less than 1.7 was re-extracted
with 1 volume of chloroform until purity >1.7 was achieved.
For blood sampling, male Sprague-Dawley rats (300-350g) were anesthetized and
the jugular vein was exposed via an incision, isolated, ligated, and nicked with
ophthalmic scissors. A sterile silatstic (0.64 cm internal diameter by 0.12 cm outer
diameter, 10 cm in length) filled with sterile saline was threaded 30-40 mm into the
jugular vein and positioned just distal to the entrance to the right atrium and secured by
6.0 silk sutures. For injections, the femoral vein was isolated, and pDNA was injected
into the femoral vein using a 27-gauge needle. This method is graphically illustrated in
Figure 3-1 and 3-2. Isolated blood samples (approx. 300 pi) were drawn through the
jugular vein cannula and immediately placed in test tubes containing 0.57 ml of 0.34 M
EDTA (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) on ice at the times indicated.
This concentration of EDTA has previously been shown to inhibit the degradation of
pDNA in isolated rat plasma (Houk 1999).

75
1
2
«-OC
«-SC
Figure 4-1 Agarose gel analysis of pDNA after conversion to the OC form of the plasmid.
Lane 1: Prior to treatment plasmid is predominately SC. Lane 2: After treatment plasmid
is completely converted to to OC form..

Spectrophotometrically calculated pDNA cone, (ug/ml)
76
3500
3000 â– 
2500 -
2000 â– 
1500 â– 
1000 â– 
500 -
0-
Supercoiled
Open Circular
Figure 4-2. Absorbance of pDNA before and after conversion to the OC form. Data
represents averages of n=3 ± 1 standard deviation.

77
1
2 3 4
Figure 4-3. Agarose gel analysis of pDNA before and after conversion to the L form of
the plasmid. Lane 1: Size standard, Lane 2: before treatment the plasmid is
predominately in the SC and OC form, Lane 3: Mixture of SC, OC, and L plasmid for
reference, Lane 4: after treatment the plasmid is completely converted to the L form.

78
To isolate pDNA from whole blood 250 pi of blood was liquid/ liquid extracted
with 250 pi of phenol: chloroform: isoamyl alcohol (25: 24: 1, v/v/v), vortexed for 5 s at
low speed, and centrifuged at 20,800 g for 10 min at room temperature. The aqueous
phase was removed and stored at -20°C until analysis. Samples analyzed and quantitated
as described in Chapter 2.
Results
SC pDNA was detectable in the bloodstream only after a 2500 pg dose, no SC
pDNA was detectable in the bloodstream at lower doses as early as 30 sec after
administration. Because of this, the pharmacokinetic parameters reported for this form of
pDNA relied only on data acquired from this dose. SC pDNA remained detectable in the
plasma through 1 min after administration. Using the limited available data we
approximated that SC pDNA degraded with a half-life of 0.15 (±0.01) min. The
degradation of SC pDNA was fit to a one-compartment body model with central
elimination and uptake (Figure 4-4). We extrapolated a least squares fit of the data to an
initial, t=0, concentration which was necessary as this area accounted for a major portion
of the AUCoo. Clearance of SC pDNA was calculated to be 390 (±10) ml/min, and
volume of distribution was 148 (±26) ml. (Table 4-1).
Concentrations of OC and L pDNA in the bloodstream after IV bolus
administration are displayed in Figure 4-5 and 4-6 respectively. Noncompartmental
analysis of all four doses is displayed in Table 4-2 for OC and Table 4-3 for L pDNA. A
decrease in terminal slope is observable with increasing dose for the OC form of the
plasmid (Figure 4-5). Clearance of the OC form of the plasmid decreased with increasing
dose (Table 4-2). Formation clearance values for the OC form of the plasmid after the

79
administration of SC pDNA ranged from 1.3 (± 0.2) to 8.3 (± 0.8) ml/min for the 2500,
and 250 pg doses respectively. Formation clearance of the L form of the pDNA
remained constant at an average of 6.7 (± 0.2) ml/min for all doses. The 250 pg dose L
concentrations close to limits of quantitation, and thus required a large amount of
extrapolation for AUC calculation. For this reason, the 250 pg dose L analysis was
excluded from the noncompartmental analysis. Corresponding plots of OC pDNA
plasma concentrations, normalized for dose, were not superimposable (Figure 4-7)
(Gibaldi 1982).
To investigate the percent of SC plasmid that becomes OC as well as the percent
OC plasmid that becomes L, we compared the AUC obtained after IV bolus
administration of the OC and L forms of plasmid independently at 2500 and 250 pg
doses. Plasma concentrations of OC pDNA obtained after administration of 2500 and
250 pg doses are displayed in Figure 4-8 and 4-9 respectively. Noncompartmental
analysis of the OC form of the plasmid at each dose is displayed in Table 4-4. Clearance
again decreased between the 250 and 2500 pg doses 8.8 (±2.4) to 1.3 (±0.2) ml/min.
Clearance also remained consistent with that observed after administration of SC pDNA
at each dose, 8.8 (± 2.4) versus 8.3 (±0.8) ml/ min at the 250 pg dose, and 1.3 (± 0.2)
versus 1.3 (± 0.2) at the 2500 pg dose. Volume of distribution of the OC form was 43
(±15) ml.
Concentrations of L pDNA after administration of 2500 and 250 pg doses of L
pDNA are presented in Figure 4-10 and 4-11 respectively. Noncompartmental analysis is

80
0.0 0.5 1.0 1.5 2.0 2.
time (min)
Figure 4-4. Concentrations of SC pDNA in the bloodstream after 2500 pg dose. SC
pDNA remained detectable through 1 minute after administration. Data points represent
averages of n=3 ± 1 standard deviation. Lines represent a least squares fit of the data
using the model described in the Methods section.

81
Table 4-1. Pharmacokinetic parameters estimated for supercoiled pDNA based upon the
fit t=0 concentration of SC pDNA
Parameter
Value
AUC (ng/pl*min)
6.4 (±0.2)
MRT(min)
0.21 (±0.02)
Cl (ml/min)
390(±10)
Vdss (ml)
148(±26)
Half-life (min)
0.15 (±0.02)
Parameters represent averages of n=3 ±1 standard deviation.

pDNA (ng/ul)
82
Figure 4-5. Concentrations of OC pDNA after IV bolus administration of: â–  2500 pg, A
500 |ug, • 333 pg, or ♦ 250 pg of SC pDNA. Data represents mean of n=3.

pDNA (ng/ul)
83
10
1
0 10 20 30 40 50 60
time (min)
Figure 4-6. Concentrations of L pDNA after IV bolus administration of: â–  2500 pg, â–²
500 p.g, • 333 pg, or ♦ 250 pg of SC pDNA. Data represents mean of n=3.

84
Table 4-2. Noncompartmental analysis of OC pDNA after IV bolus administration of SC
pDNA.
Parameter
2500 pg
500 pg
333 pg
250 pg
Dose
Dose
Dose
Dose
AUC
1200
120(±50)
59 (±3)
18 (±2)
(ng/pl*min)
(±200)
AUC % extrapolated
1 (±0.4)
9 (±4)
10 (±6)
24 (±2)
AUMC
20000
1900
400 (±20)
130(±20)
(ng/pl*min2)
(±6000)
(±1200)
MRT (min)
16(±3)
14 (±3)
6.8 (±0.4)
7.2 (±0.3)
Cl/f (ml/min)
2.1 (±0.4)
4.8 (±2.0)
5.7 (±0.3)
14 (±1)
Cl (ml/min)
1.3 (±0.2)
3.0 (±1.2)
3.5 (±0.2)
8.3 (±0.8)
Cmax (ng/pl)
49 (± 4)
13 (±4)
6.5 (±0.3)
2.2 (±0.2)
tmax (min)
1
1
0.7 (± 0.3)
0.8 (± 0.3)
Parameters represent averages of n=3 (± 1 standard deviation).

85
Table 4-3. Noncompartmental analysis of L pDNA after IV bolus administration of SC
pDNA.
Parameter
2500 pg
500 pg
333 pg
Dose
Dose
Dose
AUC
240(±40)
52 (±25)
32 (±5)
(ng/pl*min)
AUC % extrapolated
12 (±7)
15 (±5)
13 (±7)
AUMC
7500
570
300(±20)
(ng/pl*min2)
(±2700)
(±370)
MRT (min)
31 (±6)
10(±2)
9.6 (±1.7)
Cl/f (ml/min)
10.6 (±2.0)
11 (±5)
11 (±1)
Cl (ml/min)
6.5 (±1.2)
6.9 (±2.8)
6.6 (±0.9)
Cmax (ng/pi)
5.4 (± 0.6)
3.2 (±1.0)
2.4 (±0.5)
tmax (min)
22 (± 3)
5.3 (± 4.0)
6.0 (±3.6)
Parameters represent averages of n=3 (± 1 standard deviation).

ng/ul/dose
86
0.03
80
Figure 4-7. Superposition of OC pDNA concentrations normalized for dose after
administration of •: 2500 pg, ▲: 500 pg, ♦: 333 pg, or B:250 pg dose. Data represents
mean of n=3 ± 1 standard deviation.

pDNA (ng/ul)
87
0 10 20 30 40 50 60 70 80 90
time (min)
Figure 4-8. Concentrations of OC pDNA in the bloodstream after administration of OC
pDNA at a 2500 pg dose. Data represents mean of n=3 ± 1 standard deviation.

pDNA (ng/ul)
88
5
4
3
2
1
0
time (min)
Figure 4-9. Concentrations of OC pDNA in the bloodstream after administration of OC
pDNA at a 250 pg dose. Data represents mean of n=3 ± 1 standard deviation.

89
Table 4-4. Noncompartmental analysis of OC pDNA after IV bolus administration of OC
pDNA at 2500 and 250 pg doses.
Parameter
2500 pg
250 pg
AUC (ng/pl*min)
1900(±200)
30 (±9)
AUC % extrapolated
< 1
15 (±2)
AUMC (ng/pl*minJ)
50000(±5000)
220(±140)
MRT (min)
22 (±1)
6.8 (±2.3)
Cl (ml/min)
1.3 (±0.2)
8.8 (±2.4)
Vdss (ml)
29 (±3)
56 (±5)
Parameters represent averages of n=3 ± 1 standard deviation.

90
presented in Table 4-5. The data was consistent with the clearance values observed after
administration of SC pDNA, 7.6 (± 2.4) versus 6.6 (± 1.6). Volume of distribution for
the L form of the plasmid was 38 (±12) ml.
As is displayed in Figures 4-12 and 4-13, OC AUC after administration of SC
pDNA was only 64 (± 11)% and 59 (± 11) % of the AUC after administration of OC
pDNA for the 2500 and 250 pg doses respectively. The AUC of L pDNA after
administration of OC pDNA was 105 (± 23) and 95% (± 11) of the AUC of the AUC
after administration of L pDNA for the 2500 and 250 pg doses respectively (Figures 4-14
and 4-15 respectively).
Conclusions
The results of this study reveal that all forms of pDNA (SC, OC, and L) are
rapidly cleared from the circulation. Other investigators have qualitatively commented
on the rapid clearance observed after IV bolus administration of pDNA (Lew et al. 1995;
Mahato et al. 1995; Thierry et al. 1997). However, these studies have been limited to the
OC and L forms of the plasmid. The half-life of the SC topoform has been unable to be
estimated due to lack of detection (Lew et al. 1995; Thierry et al. 1997). Thierry and
coworkers (Thierry et al. 1997) utilized electrophoresis and estimated the half-life of the
OC form of the plasmid to be in the range of 10 to 20 min at a dose of 3.5 pg/g in mice.
This corresponds to a dose of approximately 1100 pg in a rat and is in reasonable
agreement with the terminal half-lives that we observed here between the 500 and 2500
pg doses. Osaka and coworkers (Osaka et al. 1996) utilized a dose of 2.25 pg/g of
linearized radiolabeled plasmid and found the half-life to be 6.6 and 11.5 min (n=2). This
corresponds to an approximate dose of 730 pg in a rat. We found the half-life of

pDNA (ng/ul)
91
Figure 4-10. Concentrations of L pDNA in the bloodstream after administration of L
pDNA at a 2500 pg dose. Data represents averages of n=3 ± 1 standard deviation.

pDNA (ng/ul)
92
4
1
0 —
0 2 4 6
time (min)
Figure 4-11. Concentrations of L pDNA in the bloodstream after administration of L
pDNA at a 250 pg dose. Data represents averages of n=3 ± 1 standard deviation

93
Table 4-5. Noncompartmental analysis of L pDNA after IV bolus administration of L
pDNA at 2500 and 250 pg doses
Parameter
2500 pg
250 pg
AUC (ng/pl*min)
330 (± 40)
22 (±4)
AUC % extrapolated
3.0 (±1.0)
51 (±16)
AUMC (ng/pl*min¿)
1500(±200)
77 (±24)
MRT (min)
4.5 (±0.1)
3.5 (±1.2)
Cl (ml/min)
7.6 (±0.8)
11 (±2)
Vdss (ml)
34 (±4)
51 (±17)
Parameters represent averages of n=3 ± 1 standard deviation.

AUC (ng/uTmin)
94
2500
2000
1500
1000
500
0
OC AUC after OC
*
OC AUC after SC
Figure 4-12. Area under the curve of OC pDNA after administration of a 2500 pg dose
of SC or OC pDNA. Data represents mean of n=3 ± 1 standard deviation. * Indicates
statistical significance by one way ANOVA (p<0.05).

AUC (ng/ul*min)
95
Figure 4-13. Area under the curve of OC pDNA after administration of a 250 pg dose of
SC or OC pDNA. Data represents mean of n=3 ± 1 standard deviation. * Indicates
statistical significance by one way ANOVA (p<0.05).

AUC (ng/ul*min)
96
500
400
300
200
100
0
L AUC after L L AUC after
OC
*
L AUC after SC
Figure 4-14. Area under the curve of L pDNA after administration of a 2500 pg dose of
SC or OC pDNA. Data represents mean of n=3 ± 1 standard deviation. * Indicates
statistical significance by one way ANOVA (p<0.05).

AUC (ng/ul*min)
97
30
25
20
15
10
5
0
L AUC after L
f
L AUC after OC
Figure 4-15. Area under the curve of L pDNA after administration of a 250 pg dose of
SC or OC pDNA. Data represents mean of n=3 ± 1 standard deviation. AUC differences
were not statistically significant by one way ANOVA.

98
linear pDNA to be 1.7 (± 0.4) in our experiments. This difference may be due to species
variation or an inability to differentiate the free radiolabel.
The SC form of the plasmid disappeared rapidly from the circulation. One
possible explanation for the rapid disappearance of SC pDNA is that it is rapidly being
converted to the OC form in vivo by endogenous plasma nucleases. Traditionally the
presence of DNase I, which is present at concentrations averaging 26.1 (± 9.2) ng/ml in
the sera of normal humans (Chitrabamrung 1981), has led to the conclusion that pDNA
administered IV is degraded rapidly [Gosse, 1965 #20; Chused, 1972 #19]. If we
compare the reported concentrations of DNase I in human plasma along with the reported
SC pDNA nicking activity of DNase I under optimal conditions (Dwyer 1999), and make
the assumption that the activity of rat DNase I is approximately the same as the human
isoform (Takeshita et al. 1996), we can arrive at an approximate activity of 0.1 (ng
pDNA/pl/min). This is far less than the in vivo SC pDNA nicking rate of 9.2 (ng
pDNA/pl/min) observed 30 sec after administration of the 2500 pg dose. One minute
after administration the in vivo rate was 2.7 ng pDNA/pl/min. Thus, the activity of
DNase I would not seem to be enough to describe the rapid conversion of SC pDNA to
the OC form. The combined effect of enzymes in addition to DNase I is also insufficient
to describe the kinetics in total. After IV bolus administration, the rate of degradation of
SC pDNA was greater than 7 times faster than in isolated rat plasma.
Furthermore, if SC were being rapidly metabolized to the OC form, the AUC of
OC pDNA after administration of SC pDNA would be nearly equal to the AUC after
administration of OC pDNA. However, the AUC of OC was only 60 (± 10) % of the

99
AUC obtained after OC, leaving -40% of the plasmid to be accounted for by entrapment,
association, or conversion to a form undetectable using this method.
Open circular pDNA was cleared less rapidly than the SC form of the plasmid.
This may be partially due to the accessibility of various nucleases. Open circular plasmid
must be nicked by endonucleases on each sister strand in the same location to generate L
pDNA. It is reasonable to assume that there are single strand nicks occurring in the OC
plasmid during its entire time course of elimination. However, it is not until a second
nick is proximal to another nick in the sister strand that degradation of the OC form is
detected. Linear pDNA would be degraded by a mechanism similar to the degradation of
the OC form, but also by exonucleases acting on the free ends of the plasmid. This
additional route of degradation would contribute to L pDNA’s more rapid clearance.
Alternatively, the mechanism for the rapid clearance of SC pDNA may be due to
physical differences between the 3 forms. Previous studies comparing SC pDNA to L
pDNA have shown that SC pDNA has stronger acidity than L pDNA (Poly 1999). This
difference is the result of the density and availability of the free phosphate groups.
Acidic groups located at the external loops of SC molecules would be available and
involved in interactions, while most of the phosphate groups localized within the SC
molecule would not interact with components in the bloodstream. OC and L pDNA
however likely expose a much higher number of available acidic functional groups (Poly
1999). These anionic charges would be located all along the pDNA molecules and allow
for multiple interactions. This decreased binding affinity of SC pDNA has been
displayed in interactions with silica (Melzak 1996) and clay minerals (Poly 1999). The
SC form of the plasmid has also been shown to interact more strongly with the

100
hydrophobic stationary phase in reversed-phase high performance liquid chromatography
(Colote 1986). This difference in exposed electrostatic groups could potentially explain
the rapid clearance of SC pDNA relative to OC and L pDNA. If L and OC pDNA
interact more strongly with plasma components than SC pDNA this may decrease their
uptake by scavenger receptors or tissues. Furthermore, this association of OC and L
pDNA with plasma components may also offer some protection from plasma nucleases.
Protection from nucleases has been displayed after adsorption to proteins and is the basis
for DNase I footprinting (Lodish 1995). This would result in SC pDNA remaining free in
the bloodstream and open to nuclease digestion. Also, the increased hydrophobicity of
SC pDNA may also lead to a greater interaction with vascular endothelia, providing an
additional clearance pathway, and explaining SC pDNA’s larger volume of distribution.
Thus, these physical differences may provide some insight into the observed
pharmacokinetic differences.
OC pDNA displayed kinetics consistent with saturable elimination. Nonlinear
elimination of OC pDNA has previously been suggested using pharmacokinetic analysis
of outflow patterns from rat perfused liver studies with radiolabeled OC pDNA (Yoshida
1996). In this study Vd increased and extraction ratio decreased as perfusion dose was
increased from 1.33 to 13.3 pg/liver. The results presented here contribute further
evidence to support this finding.
In conclusion, these results indicate that naked SC pDNA is cleared rapidly from
the rat circulation after IV bolus administration at 390 (± 50) ml/min, and has a volume
of distribution of 148 (± 26) ml. AUC analysis revealed that 60 (± 10) % of the SC
pDNA degraded to the OC form of the plasmid. The OC form of the plasmid exhibits

101
nonlinear characteristics with clearance ranging from 1.3 (± 0.2) to 8.3 (± 0.8) ml/min for
the 2500 and 250 jag doses, respectively. Vd of the OC form was 43 (± 15) ml. The
conversion of the OC form of the plasmid to the L form of the plasmid appears to be
nearly complete. The L form of the plasmid is cleared at 7.6 (± 2.4) ml/min and has a Vd
of 38 (± 12) ml.

CHAPTER 5
PHARMACOKINETIC MODELING OF PLASMID DNA AFTER IV BOLUS
ADMINISTRATION IN THE RAT
Introduction
The pharmacokinetics of any drug are best studied by simultaneous measurement
of the parent drug and all of its pharmacologically active metabolites, especially if they
all possess similar pharmacological properties (Garrett 1984). Plasmid DNA exists as
three major topoforms. The native (parent) structure of non-damaged pDNA is
supercoiled (SC). Single strand nicks in the phosphodiester backbone of the pDNA yield
an open circular (OC) form. This metabolite of SC pDNA is still associated with
significant activity (-90-100%) (Adami et al. 1998; Niven et al. 1998). Further single
strand nicks to the OC pDNA yield linear (L) pDNA, associated with a significant loss of
activity (-90%). This process is schematically illustrated in Figure 1-2.
The mathematical description of the plasma concentration-time curve of a drug
after administration yields only an equation describing elimination after a given dose.
Thus, it is difficult to predict the concentration-time profile of the drug and metabolites
after administration of varying doses. A model that quantitatively describes the
transformation of the parent drug and its metabolites as a function of dose, may permit
correlations with pharmacodynamic activities, and give insight into the mechanisms of
action (Garrett 1984).
The terms “linear” and “nonlinear” describe mathematical concepts related to a
given plasma concentration-time dataset’s dependence on administered dose (Garrett
102

103
1984). Linear differential equations describe definite properties. First, transfers from
drug to metabolite are first order. Second, multiples of dose yield the same multiple of
drug concentration in any compartment at the same time. Finally, the plasma
concentration-time curve can be described by a linear sum of exponentials.
A non-linear model does not exhibit these properties (Garrett 1984). The rate of
this type of system is not simply proportional to the plasma concentration. A well-
characterized example of this type of system is the Michaelis-Menten model of
elimination of a metabolized drug in a one-compartment model. This model is described
by the following equation:
dC_ Vmax • C
dt Km + C
Where C is the parent drug’s plasma concentration, Km is the concentration at which the
system operates at half of its maximal velocity, and Vmax is the systems maximal velocity.
In this model, drug clearance is not constant, but varies with plasma concentration.
In order to properly dose and achieve the desired levels of protein transcript it will
be necessary to clearly define the pharmacokinetic parameters involved with the
clearance of pDNA from the bloodstream. Therefore it is necessary to construct a
mathematical model to predict pDNA concentrations in vivo. We have used to the
previous information to construct this model. Noncompartmental analysis had suggested
that OC pDNA was subject to non-linear elimination. AUC analysis suggested that 60
(±10) % of the SC pDNA was being converted to the OC form of the plasmid.
Furthermore, AUC analysis also suggested complete conversion of the OC form of the
plasmid to the L form. SC pDNA was detectable only after a 2500 pg dose. Therefore
the parameters for this form of the plasmid were limited to data from this dose only.

104
These characteristics of the noncompartmental analysis were incorporated into a model
describing the elimination of pDNA from the bloodstream.
The aim of this study was to accurately model the pharmacokinetics of pDNA in a
topoform specific manner after single dose administration. This information will provide
a basis upon which pharmacokinetic/pharmacodynamic models can be constructed.
Furthermore, this model may provide useful in analyzing the kinetic effects of pDNA
delivery vehicles.
Theoretical
SC and L pDNA concentrations were fit to pseudo first-order kinetics. OC pDNA
was fit to Michelis-Menten kinetics. The degradation of L pDNA is considered to yield
fragments of heterogeneous lengths, thus these products were not included in the fitted
model. The model is presented in Figure 5-1. Based on this model the following
differential equations were derived to describe the kinetics of pDNA:
~^ = ~(k, + ku)-SC
dOC _ ^ sc _ ^max( OC) ' OC
dt ~ s' (KmloC)+OQ
dL _ ^max( OC) ' OC L
dt (Km(0c) + OC)
Where SC, OC, and L are the amounts of SC, OC, and L pDNA present at time (t)
respectively. The constant ks represents the rate constant for the degradation of SC
pDNA to OC pDNA. The constant ku represents the removal of SC pDNA from the
circulation or degradation to an undetectable form. The parameter Vmax(oc) is the
apparent maximal rate of elimination of OC pDNA from the circulation. The constant
Km(oo represents the apparent concentration at which the kinetics operate at 14 Vmax. The

105
ku
Figure 5-1. Model for pDNA clearance from the bloodstream.

106
constant kL represents the first-order rate constant for L pDNA.
The kinetics of OC pDNA after IV bolus of OC pDNA were fit to a Vmax model:
dOC _ _ ^max(OC) ' OC
dt Km(oc) + OC
The kinetics of L pDNA after IV bolus of L pDNA were fit to:
dt
Non-linear curve fitting and statistical analysis were carried out using Scientist
(version 4.0, Micromath, Salt Lake City, UT). Goodness of fit was assesses using model
selection criteria (MSC) (MicroMath 1995). Area under the plasma concentration time
curve (AUC) was calculated using trapezoidal rule. Pharmacokinetic analysis was
carried out using standard pharmacokinetic parameters as noted in the text. Statistical
analysis was performed using SAS (Version 6.12, The SAS institute, Cary, NC).
Results
The concentrations of supercoiled pDNA were detectable only after the 2500 pg
dose. The parameters for this form of the plasmid were fixed for all other doses.
Resulting observed and fitted data for the SC pDNA were presented in Figure 4-6. SC
pDNA was eliminated with a ft/2 of 0.15 (± 0.01) min.
Plasma concentrations and resulting fitted data for the OC and L pDNA are
presented in Figure 5-2 to 5-5 for the 2500, 500, 333, and 250 pg doses, respectively.
Calculated pharmacokinetic parameters are presented in Table 5-1. Predicted data agreed
well with experimental observations. Model selection criteria were 4.1, 4.3, 3.2, and 4.0
for the 2500, 500, 333, and 250 pg doses, respectively. OC pDNA was eliminated with

107
an average Vmax of 1.7 (± 0.5) ng/pl/min and an average Km of 7.1 (±2.1) ng/pl over all
doses. L pDNA was eliminated with an average ti/2 of 1.7 (± 0.5) min. Parameters
calculated after the data from all doses were fit simultaneously to the model are presented
in Table 5-2. This global fitting of the data resulted in an increase in model selection
criteria to 4.4.
We next sought to determine if the calculated pharmacokinetic parameters would
remain consistent after administration of each form of the plasmid independently versus
after administration of the SC form of the plasmid. Thus OC pDNA was administered at
2500 and 250 pg doses and the concentrations of OC and L pDNA in the bloodstream
monitored. Non-linear curve fitting for the OC form of the plasmid was carried out using
a model for saturable metabolism as described in the methods.
Predicted concentrations of OC pDNA agreed well with experimental data. Experimental
and observed data are presented in Figures 5-6 for the 2500 and 250 pg doses.
Calculated pharmacokinetic parameters are presented in Table 5-3. The elimination of
OC pDNA was more appropriately described by Michaelis-Menten elimination with a
statistical improvement of fit observed after utilization of this model. OC pDNA was
eliminated with a Vmax of 1.0 (±0.3) ng/pl/min and a Km of 3.9 (± 0.9) ng/pl. L pDNA
was eliminated with an average ti/2 of 1.7 (± 0.5) min after administration of OC pDNA
(Figure 5-7).
The L form of the plasmid was also administered at 2500 and 250 pg doses by IV
bolus administration. The concentrations of L pDNA were then monitored. Non-linear

108
A B
Figure 5-2. Concentrations of (A) OC and (B) L pDNA in the bloodstream after 2500 pg
dose of SC pDNA. Data points represent the averages of n=3 ±1 standard deviation.
Lines represent concentrations predicted by the model.

109
A B
Figure 5-3. Concentrations of (A) OC and (B) L pDNA in the bloodstream after 500 pg
dose of SC pDNA. Data points represent the averages of n=3 ±1 standard deviation.
Lines represent concentrations predicted by the model.

110
A B
Figure 5-4. Concentrations of (A) OC and (B) L pDNA in the bloodstream after 333 pg
dose of SC pDNA. Data points represent the averages of n=3 ±1 standard deviation.
Lines represent concentrations predicted by the model.

Ill
A B
Figure 5-5. Concentrations of (A) OC and (B) L pDNA in the bloodstream after 250 pg
dose of SC pDNA. Data points represent the averages of n=3 ±1 standard deviation.
Lines represent concentrations predicted by the model.

112
Table 5-1. Pharmacokinetic parameters for pDNA based upon the model presented in the
text.
Parameter
2500 pg
500 pg
333 pg
250 pg
ku (min'1)
1.9 (±0.1)
“
”
“
^(min')
2.8 (±0.2)
“
”
Vmax (ng/pl/min)
1.3 (±0.1)
2.0 (±0.4)
2.1 (±0.6)
1.3 (±0.4)
Km (ng/pl)
6.1 (±0.8)
8.8 (±3.7)
7.9 (±0.7)
5.7 (±0.6)
¿/(min’1)
0.39 (±0.02)
0.42 (±0.01)
0.42 (±0.03)
“
MSC
4.3 (±1.4)
3.4 (±1.4)
3.6 (±0.5)
3.0 (±0.6)
Parameters represent averages of n=3 ±1 standard deviation.

113
Table 5-2. Overall pharmacokinetic parameters for pDNA when all doses are fit
simultaneously.
Parameter
Value
Vmax (ng/pl/min)
1.4 (±0.1)
Km (ng/pl)
7.2 (±1.9)
ki(miril)
0.43 (±0.16)
MSC
4.4
Parameters represent simultaneously fitted values (istandard deviation of the fit) from
means of pDNA concentrations in the bloodstream after administration of SC pDNA at
2500, 500, 333, and 250 pg doses.

114
curve fitting for the L form of the plasmid was carried out using a one compartment
model with first-order elimination as described in the methods section.
Predicted concentrations of L pDNA agreed well with experimental data.
Experimental and observed data for L pDNA are presented in Figures 5-8 for the 2500
and 250 pg doses. Calculated pharmacokinetic parameters for L pDNA are presented in
Table 5-4. L pDNA was eliminated with a ti/2 of 2.15 (± 0.15) min.
Conclusions
Noncompartmental analysis had suggested several different characteristics of
pDNA pharmacokinetics. First it had suggested that the conversion of SC to OC pDNA
was not a complete process. Instead 60 % of the SC pDNA appeared to be converted to
the OC form of the plasmid. The results of the curve fitting experiments further
supported this relationship with ks able to be fit to 60 % of the overall elimination of SC
plasmid (i.e. the sum of ks and ku).
A weakness of this model is that the SC form of the plasmid was detectable only
after the 2500 pg dose. Thus, we were forced to assume linear pharmacokinetics for this
form of the plasmid. It is true that this elimination could occur more rapidly at lower
doses. However, the rate at the 2500 pg dose is already so rapid, half-life = 0.15 (±0.02)
min, that it is unlikely that fixing this parameter would significantly affect the calculated
values for the OC and L form of the plasmid.
Secondly, the noncompartmental analysis suggested non-linear processes were
involved in the elimination of the OC plasmid from the bloodstream. Curve fitting to
first order parameters also resulted in similar suggestions between doses. The value of
the first order rate constant for OC elimination ranged from 0.035/min to 0.14/min

115
A B
Figure 5-6. Concentrations of OC pDNA in the bloodstream after (A) 2500 pg and (B)
250 (ig dose of OC pDNA. Data points represent the averages of n=3 ±1 standard
deviation. Lines represent concentrations predicted by the model.

116
A
B
Figure 5-7. Concentrations of L pDNA in the bloodstream after (A) 2500 p.g and (B) 250
jag dose of OC pDNA. Data points represent the averages of n=3 ±1 standard deviation.
Lines represent concentrations predicted by the model.

117
A B
Figure 5-8. Concentrations of L pDNA in the bloodstream after (A) 2500 fag and (B) 250
fig dose of L pDNA. Data points represent the averages of n=3 ± 1 standard deviation.
Lines represent concentrations predicted by the model.

118
Table 5-3. Pharmacokinetic parameters calculated after administration of OC pDNA at
2500 and 250 pg doses.
Parameter
2500 pg Dose OC
250 pg Dose OC
Vmax (ng/pl*min)
1.1 (± 0.1)
0.90 (± 34)
Km (ng/pl)
4.4 (± 0.4)
3.4 (± 1.1)
ki (min'1)
0.39 (± 0.12)
0.49 (± 0.07)
MSC
4.4 (± 0.4)
3.4 (± 1.1)
Parameters represent mean of n=3 ± 1 standard deviation.

119
Table 5-4. Pharmacokinetic parameters calculated after administration of L pDNA at
2500 and 250 pg doses.
Parameter
2500 pg Dose L
250 pg Dose L
ki (min'1)
0.32 (± 0.02)
0.33 (± 0.03)
MSC
2.4 (± 0.6)
2.1 (± 0.9)
Parameters represent mean of n=3 ± 1 standard deviation.

120
between the 2500 and 250 pg dose data sets, respectively. Thus, it was necessary to
include non-linear elimination into the model to describe this change in elimination
between doses.
Non-linear elimination of pDNA has previously been suggested using
pharmacokinetic analysis of outflow patterns from rat perfused liver studies with
radiolabeled OC pDNA (Yoshida 1996). In this study volume of distribution decreased,
as perfusion dose was increased from 1.33 to 13.3 pg/liver, from 0.598 (±0.09) to 0.314
(±0.08) ml/g respectively. Extraction percentage decreased from 45.56 (±0.31) to 20.12
(±0.75) % as dose ranged from 1.33 to 13.3 pg/liver respectively. Thus, the results
presented here contribute further evidence to support nonlinear processes in the
elimination of OC pDNA from the circulation.
The values of Vmax and Km remained relatively constant between doses of SC
pDNA and also after administration of OC versus after administration of SC pDNA.
After administration of SC pDNA, the average of all doses for Vmax was 1.7 (± 0.5)
versus 1.0 (± 0.3) ng/pl/min after administration of OC pDNA. Likewise, the value of kL
remained relatively constant after administration of all 3 forms of the plasmid. The value
of ^ ranged from 0.47 (± 0.11) to 0.45 (± 0.10) to 0.32 (± 0.02) mifr'after administration
of SC, OC, or L pDNA respectively.
The presented data were all weighted with a factor of 0. This has resulted in some
of the lower concentration data points being less well approximated by the model than
higher concentrations. More sensitive quantitation techniques may allow the use of
weighting factors in the fitting and provide better predictions of lower concentration data.

121
Thus, we conclude from the modeling experiments that SC pDNA is rapidly converted to
the OC form of the plasmid with a half-life of 0.15 (± 0.01) min. OC pDNA exhibits
non-linear characteristics with a Vmax of 1.5 (± 0.6) ng/pl/min and Km of 6.0 (± 2.4)
ng/pl. The L form of the plasmid exhibits first-order kinetics and is eliminated with an
overall average half-life of 1.6 (± 0.4) min.

CHAPTER 6
PHARMACOKINETICS OF LIPOSOME: PLASMID DNA COMPLEXES
Introduction
One of the major obstacles to effective gene therapy is the generally poor
efficiency of pDNA delivery (Thierry et al. 1997). Most gene therapy efforts involve the
use of retroviral vectors due to their efficiency and stable integration (Thierry et al.
1997). Clinical use of retroviral vectors, is however faced with a large number of
obstacles. Among these are laborious preparation, difficulties in purification, concerns
for the recombination with endogenous virus to produce a potentially infectious virion,
and integration into the host genome to produce a tumorigenic or cytotoxic event (Liu
1999). Because of these limitations, the use of non-viral techniques, including liposomal
delivery, has become an intensely investigated area. An early published report displaying
incorporation of pDNA into liposome: pDNA complexes speculated that “...possibly
such liposomes could be used as vehicles for the introduction of new genes into cells”
(Osaka et al. 1996). This report was followed by a large number of studies demonstrating
successful in vitro liposome-mediated transgene expression in prokaryotic and eukaryotic
cells (Osaka et al. 1996). Cationic lipids have been shown to be safe and are currently
being utilized in gene therapy clinical trials (Valere 1999).
The data from the previous studies presented here, display that the circulation
time of pDNA in the circulation is remarkably short, with a rapid conversion from the SC
form of the plasmid to the OC and L forms. This change in topoform has been shown to
affect transcriptional activity (Murray 1991; Niven et al. 1998). For successful gene
122

123
therapy, longer circulation times of the more transcriptionally active SC and OC forms of
the plasmid would likely be beneficial. Furthermore, liposomal complexation with
plasmid DNA offers the advantage of conjugation of a targeting ligand. Thus this may
allow pDNA to be directed to the desired site of action.
Although few studies are available on the pharmacokinetics of liposome:pDNA
complexes, liposomal pharmacokinetics alone have been studied extensively with several
reviews published (Hwang et al. 1997; Juliano 1988; Takakura et al. 1996). Liposome
pharmacokinetics have been shown to be dependent upon size (Sato 1986), dose
(Bosworth and Hunt 1982; Osaka et al. 1996), lipid composition (Gabizon 1988), and
charge (Juliano 1988). In general, liposomes larger than 60 nm in diameter are unable to
access tissues having continuous capillary endothelia, including skeletal, cardiac, and
smooth muscle, lung, skin, subcutaneous tissue, and serous and mucous membranes, and
are limited to uptake in tissues of the reticuloendothelial system (Hwang et al. 1997).
Liposomes larger than 0.5 pm are confined to the vasculature in all tissues.
After systemic administration of liposome:pDNA complexes, pDNA rapidly
disappears from the bloodstream. Niven and coworkers (Niven et al. 1998) showed that
2.9 % of the dose of liposome:pDNA complexes could be recovered in the bloodstream 5
minutes after administration. Plasmid remained detectable (< 2% of the dose recovered)
in the bloodstream through 24 h after administration. The clearance processes involve
degradation in the blood stream (as displayed in the previous studies), interaction with
plasma proteins, organ distribution, and uptake by the reticuloendothelial system (Juliano
1988). The movement of liposomeipDNA complexes into tissues has been suggested to

124
be roughly a unidirectional system, where distribution back into the central compartment
can be assumed to be negligible (Mahato et al. 1997).
Previous studies had suggested that liposome: pDNA complexes offered
protection of the pDNA from degradation by plasma nucleases (Thierry et al. 1997), but
pDNA was removed from the circulation in a more rapid fashion than after
administration of the naked plasmid (Osaka et al. 1996; Niven et al. 1998). Niven and
coworkers (Niven et al. 1998), using [33P]pDNA, found that 36 % of the pDNA dose
could be recovered in the bloodstream at 5 min after administration of naked pDNA and
only 2.9 % of the dose could be recovered at 5 min after administration of liposome:
pDNA complexes. Osaka and coworkers (Osaka et al. 1996) also found similar results 2
minutes after administration of liposome [33P]pDNA complexes with 6.12 % of dose
equivalents/g in the blood after administration of liposome:pDNA complexes versus
15.79 % of dose equivalents/g after administration of free [33P] pDNA.
A large number of cationic lipids have been synthesized since the initial report in
1987 (Liu et al. 1997). In vitro the incorporation of the neutral phospholipid
dioleoylphosphatidylethanolamine (DOPE) into 1,2-dioleoyl-3-trimethylammonium
propane (DOTAP) liposomes helps to destabilize the endocytic vacuole membrane
allowing the release of exogenous DNA into the cytosol (Osaka et al. 1996), and
represents a commonly used lipid mixture. While in vivo, the use of DOTAP: cholesterol
liposomes is more common (Barron et al. 1998). Thus, these 2 lipid combinations were
chosen to study the effects of liposome complexation on the pharmacokinetics of pDNA.
The objective of these studies was to investigate the potential protective effects of
liposome complexation on preservation of the SC topoform. We began by initially

125
studying, in vitro, the protective effects of liposome complexation in isolated plasma.
We next analyzed the effect of liposome:pDNA ratio on this protection. Finally we
sought to compare the effects of liposome complexation on the in vivo pharmacokinetics
of pDNA with the pharmacokinetics of naked pDNA at equivalent dose.
Methods
All chemicals used were obtained similar to their description in Chapter 2
Methods. Plasmid DNA was obtianed as described in Chapter 2 Methods. All lipids
were purchased from Avanti Polar Lipids (Alabaster, AL).
For in vivo experiments, liposomes were prepared by mixing l,2-dioleoyl-3-
trimethylammonium propane (DOTAP) and cholesterol in a 1:1 molar ratio in chloroform
and drying the mixture under nitrogen at 60°C in a Buchi Rotovapor. The dried lipid film
was then reconstituted in sterile water containing 5% dextrose at a total lipid
concentration of 5 mg/ml and shaken in a 60°C water bath for 15 min. Liposomes were
then sized by sonication with an ultrasonic probe (Fisher Scientific, Springfield, NJ).
Liposome: pDNA complexes were formed by mixing liposomes with pDNA at a 2:1
lipid: DNA weight ratio and incubating at room temperature for 15 min.
For in vitro experiments, lipid: pDNA complexes were formed using 1,2-dioleoyl-
3-trimethylammonium propane (DOTAP) and dioleoyl phosphatidylethanolamine
(DOPE) in a 1:1 molar ratio. Lipid: pDNA complexes were formed by complexing
pGE150 with DOTAP: DOPE liposomes for 15 min prior to beginning the experiment.
Complexes were formed at 1:1, 3:1 and 6:1 lipid: DNA ratios (w/w). Complexes were
then incubated in rat plasma and samples drawn at various time points as described
previously. Statistical analysis was carried out using SAS (Version 6.12, SAS, Cary,
NC) and a two tailed equal variance student’s t-test.

126
For in vitro experiments, blood was isolated from male Sprague-Dawley rats
(300-350g) by cardiac puncture, and immediately placed in heparinized test tubes
(Vacutainer, Becton Dickinson, Franklin Lakes, NJ) on ice. Blood samples were
centrifuged at 6,000 g for 5 min. 600 pi of plasma was removed and placed on ice until
assay. Plasma samples were warmed to 37° C in a water bath, and maintained at 37° C
for the duration of the experiment. 12 pg of pDNA, in phosphate buffered saline, was
incubated in the 37° C plasma and 50 pi samples were taken at the times indicated. 80 pi
of phenol: chloroform: isoamyl alcohol (25: 24: 1, v/v/v) was immediately added to each
sample, vortexed for 5 s at low speed, and placed on ice. Samples were centrifuged at
20,800 g for 10 min at room temperature. From the supernatant, an aliquot of 15 pi was
removed, 5 pi of 1 x loading dye (Promega, Madison, WI) added, and placed on ice until
loaded on an agarose gel. A final volume of 10 pi was loaded on agarose gels. Analysis
and quantitation was performed as described in Chapter 2 Methods.
IV administration and analysis of liposome: pDNA complexes were performed as
described earlier in Chapter 3 methods. Briefly, SC liposome: SC pDNA complexes
were administered to male Sprague-Dawley rats (300-350 g) by IV bolus in the femoral
vein at a dose equivalent to 500 pg of pDNA. Blood samples were drawn through a
jugular vein cannula. Isolated blood samples (approximately 300 pi) were immediately
placed in tubes containing 57 pi of 0.34mM EDTA. Samples were then liquid: liquid
extracted with phenol: chloroform: isoamyl alcohol (25: 24:1 v/v/v). Quantitative
analysis was performed as described earlier in Chapter 3 methods.

127
Results
Pharmacokinetics of Liposome:pDNA Complex Degradation in Rat Plasma
To investigate the potential protective effects of cationic liposomes from plasma
nucleases, we complexed a common liposomal delivery vehicle, DOTAP: DOPE (1:1
w/w), to pDNA (3:1 lipid: DNA, w/w) and incubated the complexes in freshly isolated rat
plasma at 37°C. Figure 6-1 displays that a portion of the SC pDNA remained detectable
through 5.5 h (8.2% of the time=0 amount).
We next sought to determine the effect of lipid: pDNA ratio on the degradation
observed in the previous experiment. We hypothesized that increasing the lipid:pDNA
ratio should offer increased protection from nucleases. We chose to investigate this by
complexing lipid: pDNA at 1: 1. 3:1, and 6:1 ratios. Agarose gel analysis revealed that
the SC pDNA was detectable through at least 5 hours in all three ratios (Figure 6-2).
Quantification of the percent SC remaining revealed that 28.9%, 37.8%, and 17.7% (for
6:1, 3:1, and 1:1 respectively) of the 1 minute amount remained at 3 hours. A statistical
analysis of the percent SC plasmid remaining at revealed that there was no statistically
significant difference between the percent remaining in the 6:1 versus 1:1, 3:1 versus 1:1
and the 6:1 versus 3:1 ratios. These results suggest increasing the lipid: DNA ratio from
1:1 to 6:1 offers no significant increase in protection from plasma nucleases through 3
hours.
The OC and L forms of the plasmid were hypothesized to appear for three
possible reasons. First, it is possible that some portion of the pDNA remains non-
complexed and free in solution. When this mixture of complexed pDNA and free pDNA
is incubated in the plasma, the free pDNA degrades as previously described, and thus the
OC and L pDNA appear and degrade. A second possible explanation is that the pDNA

128
on the outer surface of the liposome: pDNA complex aggregates is offered little
protection from plasma nucleases and is degraded. The pDNA towards the core of the
complex is then spared from degradation. Finally, it is possible that the complexes
degrade in the plasma.
To confirm that all pDNA was complexed, we complexed lipid: pDNA at 1:1, 3:1,
and 6:1 ratios (w/w) and analyzed their migration through an agarose gel. If any pDNA
remained non-complexed, it should migrate through the gel and separate from the
complexed pDNA remaining in the well. No migration from the wells was observed at
any lipid: pDNA ratio as seen in Figure 6-3. These results suggest that all pDNA is
complexed at all three ratios, pDNA must therefore be degrading from the surface of the
liposomes or the complexes must dissociate in the plasma.
Pharmacokinetics of Liposome:pDNA Complex Degradation in Rat Whole Blood
We next sought to determine if the effects we observed using liposomes in the
plasma were consistent with the effects we would observe in whole blood. Unlike naked
pDNA, liposome/pDNA complexes have been shown to be recovered in the red blood
cells (37 to 84 %) (Osaka et al. 1996). We hypothesized that this uptake may affect the
kinetics observed. Liposome/pDNA complexes taken up by the red blood cells may be
may be disassembled in the RBC and pDNA degraded when freed from the protective
liposome complex. Thus our hypothesis was that pDNA would break down faster in
whole blood. We also sought to determine the effects of increasing the lipid:pDNA ratio
on this process, and secondarily hypothesized that increasing the lipid:pDNA ratio should
lead to more rapid uptake by RBC, resulting in more rapid degradation of the pDNA.
The results of the whole blood experiments revealed that the protocol used in
isolation of pDNA from the plasma also were sufficient to isolate pDNA from whole

129
blood. Plasmid DNA incubated in whole blood did not degrade faster than pDNA in the
plasma (Figure 6-4 A). Furthermore, increasing the lipid:pDNA ratio from 3:1 to 6:1 did
not serve to increase the degradation observed (Figure 6-4 B).
These results suggest that the presence of RBC does not significantly affect the
degradation rate observed. An increase in lipid:pDNA ratio also does not affect the
degradation observed in the plasma. Liposome complexation results in a relative
maintenance of the SC topoform with 28.9%, 37.8%, and 17.7% (for 6:1, 3:1, and 1:1
respectively) of the 1 minute amount remaining after 3 hours.
Pharmacokinetics of Liposome:pDNA Complexes after IV Bolus Administration in the
Rat
The in vitro results displayed that liposome complexation can protect a portion of
the SC topoform from degradation through 5.5 hours. We next sought to determine if
liposome complexation would provide similar protection in vivo. Thus, we injected
liposome: SC pDNA complexes at a 500 pg dose of pDNA as described in the methods
section.
Agarose gel analysis obtained after administration of liposome: pDNA complexes
is presented in Figure 6-5. Plasma concentrations of SC, OC, and L pDNA are presented
in Figure 6-6. Unlike after administration of naked pDNA, SC pDNA was readily
detectable after administration, and remained detectable through 5 minutes after
administration of the 500 pg dose. However, the OC and L form of the plasmid also only
remained detectable through 5 min after administration, versus through 20 minutes after
administration of naked pDNA.
Noncompartmental analysis of all three forms of the plasmid is presented in Table
6-1, and a relative comparison of the naked and liposome complex parameters is

130
presented in Tables 6-2, 6-3, and 6-4 for SC, OC, and L pDNA, respectively. If we again
make the assumption that SC pDNA follows linear pharmacokinetics, mean residence
time for the SC form of the plasmid increased from 0.21 (± 0.02) after administration of
naked pDNA to 0.99 (± 0.42) min after administration of liposome:SC pDNA complexes.
Clearance decreased from 390 (± 10) to 87 (± 30) ml/min. Obviously this disparity
would only become more severe if the true pharmacokinetics of naked SC pDNA are
more rapid than this at the 500 pg dose. Area under the concentration time curve of the
OC form of the plasmid decreased after liposome complexation from 120 (±50) to 14
(±4) ng/pl*min. Clearance/f of the OC form of the plasmid increased from 6.9 (±2.8) to
95 (±37) ml/min. Area under the concentration time curve (AUC) of L pDNA also
decreased from 52 (±25) to 5.7 (±1.9) ng/pl*min.
Conclusions
These results indicate that liposome complexation can indeed offer protection of
the SC form of the plasmid. This increase in circulation half-life provides further
evidence to support the use of liposomal delivery vehicles in gene therapy. This analysis
also provides evidence that, although the liposome complexation does protect the SC
form of the plasmid, the individual particles have an overall more rapid clearance from
the circulation. The OC and L form of the plasmid also only remained detectable through
5 min after administration, versus through 20 minutes after administration of naked
pDNA.
This finding was mirrored by the [33P]DNA results of Niven and coworkers
(Niven et al. 1998) who found that 36 % of the pDNA dose could be recovered in the
bloodstream at 5 min after administration of naked pDNA and only 2.9 % of the dose

131
Figure 6-1. Liposome-pDNA complexes were incubated in rat plasma for various time
points. 10 pi of sample was loaded in each lane as described in the methods section.
Lane 1; size standard, lane 2; 1 min, lane 3; 2 min, lane 4; 5 min, lane 5; 10 min, lane 6;
20 min, lane7; 30 min, lane 8; 60 min, lane9; 2 h, lane 10; 3 h, lane 11; 5.5 h.

132
*
Figure 6-2. Agarose gel analysis of liposome/pDNA complexes. (A) 1:1 lipid:pDNA
ratio, through 4 hours. (B) 3:1 lipid:pDNA ratio, through 6 hours. (C) 6:1 lipid:pDNA
ratio, through 6 hours. “"Indicates the 3 hour time point.

133
Figure 6-3. Lane 1: high molecular weight size standard, lane 2: 1:1 lipid:pDNA
complexes, lane 3: 3:1 lipid:pDNA ratio (w/w), lane 4: 6:1 lipid:pDNA ratio

ng SC pDNA/ u|
134
0 50 10 15
0 0
time
(min)
0
time
(min)
10
0
15
0
Figure 6-4. (A)Degradation of SC pDNA in rat blood versus plasma. (B)Degradation of
supercoiled pDNA in 3:1 and 6:1 (w/w) liposome/pDNA complexes incubated in
heparinized rat whole blood. Error bars indicate standard deviation of n=3 rats.

135
1
2 3
4 5 6 7 8 9 10
Figure 6-5. Agarose gel analysis of pDNA after administration of liposome: pDNA
complexes. Lane 1:15 sec, lane 2: 30 sec, lane 3: 45 sec, lane 4: 1 min, lane 5: 1.5 min,
lane 6: 2 min, lane 7: 2.5 min, lane 8: 3 min, lane 9: 4 min, lane 10: 5 min.

pDNA (ng/ul)
136
10
Figure 6-6. Plasma concentrations of SC, OC, and L pDNA after 500 pg IV bolus
administration of SC pDNA: liposome complexes. Key: ♦: SC, ■: OC, ▲: L.

137
Table 6-1. Noncompartmental analysis of pDNA after administration of liposome: SC
pDNA complexes.
Parameter
Supercoiled
Open Circular
Linear
AUC (ng/pl*min)
0.0063 (± 0.0023)
0.014 (± 0.004)
0.0057 (± 0.0019)
AUC %
extrapolated
12 (±3)
25 (± 18)
68 (± 26)
AUMC
(ng/pl*min2)
0.0068 (± 0.0053)
0.04 (± 0.004)
0.017 (± 0.003)
MRT (min)
0.99 (± 0.42)
3.0 (± 0.9)
3.3 (± 1.0)
Cl/f (ml/min)
87 (± 30)
37 (± 9)
95 (± 37)
Parameters represent averages of n=3 ± 1 standard deviation.

138
Table 6-2. Comparison of SC pDNA pharmacokinetic parameters after administration of
SC pDNA either in free form (naked) at 2500 pg dose or after administration as
liposome: pDNA complexes at 500 pg dose.
Parameter
Naked SC pDNA
Liposome: SC pDNA
complexes
MRT (min)
0.21 (+0.02)
0.99 (± 0.42)
Cl (ml/min)
390(±10)
87 (± 30)
Vdss (ml)
148(±26)
79 (± 16)
Parameters represent averages of n=3 ± 1 standard deviation.

139
Table 6-3. Comparison of OC pDNA pharmacokinetic parameters after administration of
SC pDNA either in free form (naked) or after administration as liposome: pDNA
complexes at 500 pg pDNA dose.
Parameter
OC pDNA after Naked SC
pDNA
OC pDNA after
LiposomerSC pDNA
Complexes
AUC (ng/pl*min)
120(±50)
14 (± 4)
AUC % extrapolated
9 (±4)
25 (± 18)
AUMC (ng/pPmin2)
1900(±1200)
40 (± 4)
MRT (min)
15(±4)
3.0 (± 0.9)
Cl/f (ml/min)
3.0 (±1.2)
37 (± 9)
Cmax (ng/pl)
13 (±4)
5.8 (± 0.7)
Tmax (min)
1 (±0)
0.25 (±0)
Parameters represent averages of n=3 ± 1 standard deviation.

140
Table 6-4. Comparison of L pDNA pharmacokinetic parameters after administration of
SC pDNA either in free form (naked) or after administration as liposome: pDNA
complexes at 500 pg pDNA dose.
Parameter
L pDNA after naked SC
pDNA
L pDNA after Liposome:
SC pDNA complexes
AUC (ng/pl*min)
52 (±25)
5.7 (± 1.9)
AUC % extrapolated
15 (±5)
68 (± 26)
AUMC (ng/pPmin^)
570(±370)
17 (±3)
MRT (min)
10 (±2)
3.3 (± 1.0)
Cl/f (ml/min)
6.9 (±2.8)
95 (± 37)
Cmax (ng/pl)
3.2 (±1.0)
1.8 (± 0.8)
Tmax (min)
5.3 (±4.0)
0.25 (±0)
Parameters represent averages of n=3 (± 1 standard deviation).

141
could be recovered at 5 min after administration of liposome: DNA complexes. Osaka
and coworkers (Osaka et al. 1996) also found similar results 2 minutes after
administration of liposome [33P]DNA complexes with 6.12 % of dose equivalents/g in the
blood versus 15.79 % of dose equivalents/g after administration of free [33P] DNA.
Thierry and coworkers (Thierry et al. 1997) utilized lipospermine (DOGS) and
DOPE liposomes and also were able to display protection of pDNA from degradation. In
this study SC pDNA was detected for up to 60 min after incubation in isolated plasma,
but was rapidly eliminated when IV delivered. Thierry estimated the plasma half-life of
the OC pDNA to be much longer than the results presented here at 10 to 20 minutes.
Their work did however, show for the first time, the presence of intact pDNA in plasma
and blood cells following systemic administration. Lew and coworkers (Lew et al. 1995)
used l,2-dimyristoyl-oxypropyl-3-dimethyl ammonium bromide: DOPE liposomes and
determined a half-life of a few minutes for the OC form of the pDNA, similar to the
results presented here. However, unlike the results presented here, they reported no
detectable SC pDNA. It may be that cationic liposome: pDNA complexes may bind to
serum components, and these interactions may differ with different lipids. Indeed,
altering lipids and lipid to lipid weight ratios does affect transfection levels of various
organs in vivo (Liu 1997; Song et al. 1997).
Liu and coworkers (Liu et al. 1997) showed that higher cationic lipid to pDNA
ratio was essential to achieve better gene delivery efficiency in vivo. The results
presented in our studies, however, displayed that increasing the lipid to pDNA ratio from
1:1 to 6:1 had no effect on the observed protection from plasma nucleases in isolated

142
plasma or whole blood. However, analysis of Liu and coworkers results display that
transfection activity remained relatively stable between the 1:1 and 6:1 ratios. Ratios
higher than 24:1 were essential to achieve significant increases in transfection activity.
Thus, even higher ratios of lipid: pDNA may provide protection from plasma nucleases.
Alternatively, one must also consider the dilution effect in the bloodstream after
administration of such a necessarily large volume at these higher lipid: pDNA ratios. It is
likely that mouse vasculature is occluded with these high volumes. Thus, complexes
travel in the vasculature with little mixing with blood. This would obviously provide
protection of the pDNA. However, given that these volumes on a weight basis would not
be used in clinical trails in humans (Valere 1999), it is unlikely that this vascular
occlusion effect would be so pronounced.
Furthermore, there is likely a maximal amount of complex that can be taken up by
tissues. If there is a maximal amount that can be taken up, then there would be a
saturation lipid dose that, above which, no further increases in transfection would be
observed. This, in fact, was observed in Song and coworkers (Song et al. 1997) and Liu
and coworkers (Liu et al. 1997) experiments. Song and coworkers found that ratios from
36:1 to 48:1 offered no increase in transfection, while increases from 2:1 to 36:1 offered
significant increases. Liu and coworkers found that ratios between 24:1 and 48:1 also
offered no increase in transfection, while increasing the ratio from 6:1 to 24:1 offered
significant increases. Thus, these extremely high ratios of lipid: pDNA may be
unnecessary to provide adequate protection of the pDNA.
The OC and L pDNA were cleared from the circulation more rapidly than after
administration as naked pDNA. This may be due to the fact that liposome pDNA

143
complexes are more rapidly cleared from the circulation than naked pDNA, regardless of
the pDNA form. If this were true, one would predict that administration of radiolabeled
pDNA as liposome: pDNA complexes should show higher and more rapid increases of
tissue radioactivity than after administration as naked pDNA. This results has been
observed after administration of [33P] pDNA (Osaka et al. 1996; Liu 1997; Song et al.
1997; Niven et al. 1998). This was evident as increases in AUC maximal radioactivity
recovery/ tissue weight. Blood radioactivity exhibited larger AUCs’ and CmaXs’ after
administration of naked pDNA. This provides further evidence to support this
hypothesis.
In conclusion, liposome pDNA complexes are eliminated from the circulation
more rapidly than naked pDNA, while providing protection of the SC topoform from
degradation in the bloodstream. This level of protection is independent of lipid: pDNA
ratio from 1:1 through 6:1. SC pDNA is detectable through 5 min after administration as
liposome: pDNA complexes at a 500 pg dose, whereas it is undetectable after
administration as naked pDNA at this dose. Clearance of the SC pDNA decreased from
390 (± 10) to 87 (± 30) after administration as naked or liposome complexes,
respectively. Volume of distribution decreased from 148 (± 26) to 79 (± 16) ml after
administration as naked or liposome complexes, respectively. OC and L pDNA exhibited
decreases in Cmax after administration as liposome: SC pDNA complexes from 13 (± 4) to
5.8 (± 0.7) ng/pl and 3.2 (± 1.0) to 1.8 (± 0.8) ng/pl, respectively. Decreases in W after
administration as liposome: SC pDNA complexes were also displayed for both OC and L
pDNA from 1 (±0) to 0.25(±0) and 5.3 (+4.0) to 0.25 (± 0) min, respectively. Clearance/f
increased after administration as liposome: SC pDNA complexes for both the OC and L

144
forms of the plasmid from 3.0 (± 1.2) to 37 (± 9) and 6.9 (± 2.8) to 95 (± 37) ml/min,
respectively. Thus, liposome pDNA complexes are eliminated from the circulation more
rapidly than the naked pDNA, while providing protection of the SC topoform from
degradation in the bloodstream.

CHAPTER 7
CONCLUSIONS AND IMPLICATIONS
Summary of Results
Implications of Plasmid DNA Degradation in Isolated Plasma
DNase I is a well-characterized enzyme in human plasma present at
concentrations averaging 26.1 (±9.2) ng/ml in the sera of healthy humans (Chitrabamrung
1981). Traditionally the presence of this enzyme has led to the conclusion that pDNA
administered IV is degraded in a rapid fashion (Gosse et al. 1965; Chused 1972). This
has led to the current view of gene delivery, in which protection from plasma nucleases is
a major goal of delivery vehicles. The results of this study reveal that although the half-
life of SC and OC pDNA is remarkably short, degradation alone was not enough to
explain the rapid disappearance of pDNA from the circulation observed in vivo. After IV
bolus the rate of degradation of SC pDNA was greater than 7 times faster than in isolated
plasma.
Previous reports on the pharmacokinetics of pDNA have only been qualitative, or
involved radiolabeling. These studies indicated that pDNA degrades within 5 minutes
after incubation in whole blood in vitro or after IV injection in mice (Kawabata et al.
1995; Thierry et al. 1997). We sought to quantitatively model the pharmacokinetics
underlying the stability of pDNA in the plasma using isolated rat plasma as a model
system.
The results presented in Chapter 2 revealed that SC pDNA degrades in isolated
plasma with a half-life of 1.2 min. Open circular pDNA is more stable than the
145

146
supercoiled topoform degrading with a half-life of 21 min. Linear pDNA is degraded
more rapidly than the OC topoform with a half-life of 11 min. A schematic
representation of the kinetics of pDNA degradation in isolated rat plasma is presented in
Figure 7-1.
Liposome complexation revealed a relative maintenance of the SC topoform
through 5.5 h. This provides further evidence to suggest that liposome complexation may
not only be a means by which to deliver pDNA to target sights, but also to specifically
protect SC pDNA from degradation.
In summary the in vitro work presents a pharmacokinetic model describing the
degradation of pDNA in rat plasma. Using the model derived, we are able to conclude
that naked supercoiled pDNA degrades in rat plasma with a half-life of 1.2 (±0.1) min,
open circular with a half-life of 21 (± 1) min, and linear pDNA with a half-life of 11 (± 2)
min. Furthermore, these studies provide evidence that supercoiled pDNA can remain
stable in the plasma through 5.5 hours when complexed to cationic liposomes. This
degradation was independent of sequence between the pGL3, pGE150 and pGeneMax-
Luciferase plasmids.
Comparison of In Vitro and In Vivo Pharmacokinetics
The results presented in Chapter 3 indicate that SC pDNA was undetectable after
IV bolus administration of a 500 pg dose, whereas SC pDNA was readily detectable in
isolated plasma, and remained detectable through 3 min of incubation. Similar results
were seen for the OC and L forms of the plasmid. The terminal half-lives of OC and L
pDNA decreased from 21 (±1) to 5.3 (±1.4) and 11 (±2) to 1.9 (±0.8) min, respectively.
This indicates that nuclease activity alone is not sufficient to describe the rapid clearance.

147
0000
ks: 0.59
(±0.03) min'
I
o
l
k0: 0.033
(±0.002) min'1
SC
oc

148
of pDNA from the bloodstream in rats
Chused and coworkers (Chused 1972) also suggested that nuclease activity was
not enough to explain the rapid clearance of KB cell DNA from the circulation in mice.
In this study, only 2 to 3 % of the radioactivity was hydrolyzed to trichloroacetic acid
(TCA) soluble fragments in 30 min, which was several half-lives of the DNA in the
circulation. Tsumita and Iwanga (Tsumita and Iwanga 1963) also found that less than 5
% of the total radioactivity was found in the TCA soluble fraction after 4.5 hours in
mouse serum.
Alternatively, Gosse and coworkers (Gosse et al. 1965) suggested a major role for
nucleases in the initial degradation of DNA after IV administration in rabbits and mice.
This finding was based upon the proportionality between the initial rate of
depolymerization and the plasma DNase activity level. Also, a rapid decrease in
viscosity of isolated blood was discovered indicating a depolymerization of DNA.
Finally a markedly slower disappearance of DNA-methyl green complex (a non-specific
DNase inhibitor) than after native DNA.
The reason for this disparity in results deserved further investigation. Gosse
utilized much higher doses of pDNA in their investigations, 200 pg versus 5 pg pDNA in
Chused and coworkers ’s investigations. This disparity may be due to saturation of a
scavenger receptor, allowing nuclease activity to become increasingly important. The
effect of increasing dose on the clearance of DNA deserved further investigation.
Effects of Increasing Dose of Plasmid DNA
The results presented in Chapter 4 reveal that all forms of pDNA (SC, OC, and L)
are rapidly cleared from the circulation. Other investigators have qualitatively

149
commented on the rapid clearance observed after IV bolus administration of pDNA (Lew
et al. 1995; Mahato et al. 1997; Thierry et al. 1997). However, these studies have been
limited to the OC and L forms of the plasmid. The half-life of the SC topoform has been
unable to be estimated due to lack of detection (Lew et al. 1995; Thierry et al. 1997).
The SC form of the plasmid was detectable only after the 2500 pg dose, and
disappeared from the circulation after 1 min. One possible explanation for the rapid
disappearance of SC pDNA is that it is rapidly being converted to the OC form in vivo by
endogenous nucleases present in the plasma. If we compare the reported concentrations
of DNase I in human plasma along with the reported SC pDNA nicking activity of DNase
I under optimal conditions (Dwyer 1999) and make the assumption that the activity of rat
DNase I is approximately the same as the human isoform (Takeshita et al. 1996), we can
arrive at an approximate activity of 0.1 ng pDNA/pl/min. This is far less than the in vivo
SC pDNA nicking rate of 9.2 ng pDNA/pl/min observed 30 sec after administration of
the 2500 pg dose. One minute after administration the in vivo rate was 2.7 ng
pDNA/pl/min. Thus, the activity of DNase I would not seem to be enough to describe
the rapid conversion of SC pDNA to the OC form. The combined effect of enzymes in
addition to DNase I is also insufficient to describe the kinetics in total. After IV bolus
administration, the rate of degradation of SC pDNA was greater than 7 times faster than
in isolated rat plasma.
The mechanism for the rapid clearance of SC pDNA may also be due to physical
differences between the 3 forms. Previous studies comparing SC pDNA to L pDNA have
shown that SC pDNA has stronger acidity in solution than L pDNA (Poly 1999). This
difference is the result of the density and availability of the free phosphate groups.

150
Acidic phosphate groups located at the external loops of SC molecules would be
available and involved in interactions, while most of the phosphate groups localized
within the SC molecule would not interact with components in the bloodstream. OC and
L pDNA however likely expose a much higher number of available acidic functional
groups (Poly 1999). These anionic charges would be located all along the pDNA
molecules and allow for multiple interactions. This decreased binding affinity of SC
pDNA has been displayed in interactions with silica (Melzak 1996) and clay minerals
(Poly 1999). The SC form of the plasmid has also been shown to interact more strongly
with the hydrophobic stationary phase in reversed-phase high performance liquid
chromatography (Colote 1986). This difference in exposed electrostatic groups could
potentially explain the rapid clearance of SC pDNA relative to OC and L pDNA. If L
and OC pDNA interact more strongly with plasma components than SC pDNA this may
decrease their uptake by scavenger receptors or tissues. Furthermore, this association of
OC and L pDNA with plasma components may also offer some protection from plasma
nucleases. Protection from nucleases has been displayed after adsorption to proteins and
is the basis for DNase I footprinting (Lodish 1995). This would result in SC pDNA
remaining free in the bloodstream and open to nuclease digestion. Also, the increased
hydrophobicity of SC pDNA may also lead to a greater interaction with vascular
endothelia, providing an additional clearance pathway, and explaining SC pDNA’s larger
volume of distribution. Thus, these physical differences may provide some insight into
the observed pharmacokinetic differences.
The OC pDNA displayed kinetics consistent with saturable elimination. Curve
fitting to first order parameters also resulted in similar suggestions between doses. The

151
value of the first order rate constant for OC elimination ranged from 0.035/min to
0.14/min between the 2500 and 250 jag dose data sets, respectively. Thus it was
necessary to include non-linear elimination into the model to describe this change in
elimination between doses.
In conclusion, these results indicate that naked SC pDNA is cleared rapidly from
the rat circulation after IV bolus administration at 390 (± 50) ml/min, and has a volume
of distribution of 148 (± 26) ml. AUC analysis revealed that 60 (± 10) % of the SC
pDNA appeared as the OC form of the plasmid. The OC form of the plasmid exhibits
nonlinear characteristics with clearance ranging from 1.3 (± 0.2) to 8.3 (± 0.8) ml/min for
the 2500 and 250 pg doses, respectively. Volume of distribution of the OC form was 43
(± 15) ml. The conversion of the OC form of the plasmid to the L form of the plasmid
appears to be nearly complete. The L form of the plasmid is cleared at 7.6 (± 2.4) ml/min
and has a volume of distribution of 38 (± 12) ml.
Results of the Curve Fitting Experiments
The model presented in Figure 5-1 successfully described the data. The values of
Vmax and Km for OC pDNA remained relatively constant between doses of SC pDNA and
also after administration of OC versus after administration of SC pDNA. After
administration of SC pDNA, the average of all doses for Vmax was 1.7 (± 0.5) versus 1.0
(± 0.3) ng/pl/min after administration of OC pDNA. Likewise, the value of kL remained
relatively constant after administration of all 3 forms of the plasmid. The value of kL
ranged from 0.47 (± 0.11) to 0.45 (± 0.10) to 0.32 (± 0.02) min'1 after administration of
SC, OC, or L pDNA respectively.

152
Thus, we conclude from the modeling experiments that SC pDNA is rapidly
converted to the OC form of the plasmid with a half-life of 0.15 (± 0.01) min. OC pDNA
exhibits non-linear characteristics with a Vmax of 1.5 (± 0.6) ng/pl/min and Km of 6.0 (±
2.4) ng/pl. The L form of the plasmid exhibits first-order kinetics and is eliminated with
an overall average half-life of 1.6 (± 0.4) min. Schematic representationof pDNA
pharmacokinetic parameters after IV bolus administration of SC pDNA is presented in
Figure 7-2.
Liposome: pDNA Complex Conclusions
These results indicate liposome complexation can indeed offer protection of the
SC form of the plasmid. This increase in circulation half-life provides further evidence to
support the use of liposomal delivery vehicles in gene therapy. This analysis also
provides evidence that, although the liposome complexation does protect the SC form of
the plasmid, the individual particles have an overall more rapid clearance from the
circulation. The OC and L form of the plasmid also only remained detectable through 5
min after administration, versus through 20 minutes after administration of naked pDNA.
Liposome pDNA complexes were eliminated from the circulation more rapidly
than the naked pDNA, while providing protection of the SC topoform from degradation
in the bloodstream. This level of protection is independent of lipid: pDNA ratio from 1:1
through 6:1. SC pDNA is detectable through 5 min after administration as liposome:
pDNA complexes at a 500 pg dose, whereas it is undetectable after administration as
naked pDNA at this dose. Clearance of the SC pDNA decreased from 390 (± 10) to 87

153
(± 30) after administration as naked or liposome complexes, respectively. Volume of
distribution decreased from 148 (± 26) to 79 (± 16) ml after administration as naked or

154
-i
1.9 (± 0.1)min
Vmax 1.5 (±0.6) ng/|j.l/min
_ _ ,, _ . .i Km 6.1 (±2.4) ng/^il ,
2.8 (±0.2) min ^ ^ 0.42 (±0.1 l)mml
Figure 7-2. Schematic representation of pDNA pharmacokinetic parameters after IV
bolus administration of SC pDNA in the rat.

155
liposome complexes, respectively. OC and L pDNA exhibited decreases in Cmax after
administration as liposome: SC pDNA complexes from 13 (± 4) to 5.8 (± 0.7) ng/pl and
3.2 (± 1.0) to 1.8 (± 0.8) ng/pl, respectively. Decreases in tmax after administration as
liposome: SC pDNA complexes were also displayed for both OC and L pDNA from 1
(±0) to 0.25(±0) and 5.3 (±4.0) to 0.25 (± 0) min, respectively. Clearance/f increased
after administration as liposome: SC pDNA complexes for both the OC and L forms of
the plasmid from 3.0 (± 1.2) to 37 (± 9) and 6.9 (± 2.8) to 95 (± 37) ml/min, respectively.
These results indicate that liposome pDNA complexes are eliminated from the
circulation more rapidly than the naked pDNA, while providing protection of the SC
topoform from degradation in the bloodstream. This theory is schematically represented
in Figure 7-3. This results presented here are consistent with previously published results
(Osaka et al. 1996; Niven et al. 1998). Thus, liposome complexation may be an attractive
means by which to protect the SC topoform, but the complex ability to target specific
organs may be limited by their rapid clearance from the circulation.
Future Directions
The results and models presented here successfully described the
pharamcokinetics of pDNA after IV administration in the rat. However, there are still a
large number of studies that need to be performed. Among these directions, an important
area is to link the PK model presented here to pharmacodynamic (PD) effect. Selection
of an appropriate PD effect is not as direct of a relationship as for traditional drugs
exhibiting receptor mediated effects.

156
Figure 7-3. Schematic representation of liposome pDNA clearance from the
bloodstream. In this model, removal from the bloodstream of the lipid: pDNA complexes
is assumed to be larger than the degradation of the complex.

157
The most direct mesure of the dose-response-over time relationship between
pDNA and PD may be to measue the levels of mRNA transcript. This analysis could be
performed by quantitative polymerase chain reaction. Using this method, levels of
transcribed mRNA in various tissues could be calcualted over time and correlated to the
PK model presented here.
Alternatively, one could model the levels of transcribed protein in tissues over
time as a PD parameter. This could be done by a number of methods including enzyme
linked immunosorbent assay, western blotting, radio immuno assay, or
immunoprecipitation. This measurement is a more direct relationship to clinical
response. However, this relationship may be more difficult to obtain given that the
translation into a final protein is a multistep process. It is likely that pDNA, mRNA, and
protein stability in the cytosol differs between cells of various tissues and between cell
types of a given tissue. Thus, correlation of protein levels may be more difficult to
achieve. The kinetic processes within a cell will be an important area to be understood,
in addition to kinetic processes in the bloodstream.
Furthermore, the application of this model may also need to be adjusted for
different species. The clearance of DNA has been shown to be similar between strains of
New Zealand, DBA/2, and BALB/c mice (Chused 1972), but differed between mice and
rabbits (Gosse et al. 1965). Thus, this model may need to be adjusted not only for
parameter values, but also in necessary parameters to describe the data.
Concluding Remarks
In conclusion, this work presents a pharmacokinetic analysis of pDNA in vitro, in
isolated rat plasma, and in vivo, after IV bolus administration in the rat. Comparison of

158
the in vitro and and in vivo results displayed that degradation by plasma nucleases was
not sufficient to describe the pharmacokinetics of pDNA. In addition, the
pharmacokinetic effects of liposome complexation were investigated in vitro and in vivo.
These results displayed that liposome complexation was a means by which the SC
topoform could be protected, but the complexes had an overall more rapid clearance from
the bloodstream in vivo.

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BIOGRAPHICAL SKETCH
Brett Edward Houk was bom on April 17th 1969 in Parma Ohio, and spent most
of his childhood in Strongsville, Ohio. Brett attended the Ohio State University where he
obtained Bachelor of Science degrees in both allied medicine and economics. After
graduating from undergraduate studies, Brett moved to West Palm Beach, Florida and
worked in Lantana Public Health Clinic in clinical pharmacokinetics. It was here that
Brett decided to pursue a career as a pharmaceutical scientist. Brett was admitted to the
University of Florida College of Pharmacy as a graduate student in August of 1996.
In his spare time Brett enjoys reading, swimming, working out, and playing sports
of all varieties.

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Jeffrey A. Hughes, Chair
associate Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Guenther Hochhaus, Cochair
Associate Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
U Qw
Hartmut Derendorf
Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Associate Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
—-
Edwin M. Meyer
Associate Professor of Physiology and
Pharmacology

This dissertation was submitted to the Graduate Faculty of the College of
Pharmacy and to the Graduate School and was accepted as partial fulfillment.
requirements for the degree of Doctor of Philosophy/.
May 2000
armacy
Dean, Graduate School

UNIVERSITY OF FLORIDA
3 1262 08555 2742




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